Monitoring differentiation of HESCs in vitro by RT-PCR. Relative gene expression is shown as fold change in gene expression using the comparative Ct (ΔΔCt) method. Expression levels of genes in the initial HESC sample were used as standard (equal to 1) to which all other expression values were compared and expressed as fold changes. Bars corresponding to data from P2 NPs are shown in darker colors, to distinguish them from data derived from day 7-28 HESC colonies that continuously differentiate within the same plates.
A. Expression of index pluripotency and neural markers in differentiating HESCs, measured by quantitative RT-PCR at day 7, 14, 21, 28 and 49 (P2). Stem cell markers are shown in yellow, early neural markers are shown in green, neuronal and glial markers are in grey, neural crest markers are shown in blue, and endodermal markers are in pink. The expression of mesodermal marker Brachyury was not detectable and, therefore, is not shown.
B. Expression of trophic factors including neurotrophins (Nerve Growth Factor, NGF and Brain-Derived Neurotrophic factor, BDNF), Glial-Derived Neurotrophic Factor (GDNF), Vascular Endothelial Growth Factor (VEGF) and basic fibroblast growth factor (bFGF) by differentiating HESCs at day 7, 14, 21, 28 and 49.
C. Expression of selected dorsal-ventral (blue), anterior (pink) and posterior (purple red) markers by differentiating NPs at day 28 and P2. Several markers (Otx2, Emx2, Dlx2 and Nkx2.1) were not detectable and therefore are not shown.

Transplantation strategy and survival of human NPs in rat neostriatum.
A. This is a composite sagittal map of individual striatal grafts at maximal inoculation size. Contours of each individual graft are shown in black. Contours of rat brain and the ventricular lining are in bold grey. The map shows a reliable coverage of a large section of anterior caudate-putamen with rare invasion into the adjacent globus pallidus. Major anatomical landmarks are depicted for orientation purposes (see abbreviations below).
B. This coronal section through the anterior septum shows a typical striatal graft of human NPs 3 months after inoculation.
C. This is an HNu-stained sagittal section through the middle of HESC-derived NP graft 3 months after grafting. Section was counterstained with cresyl violet. Most HNu (+) cells have large round nuclei consistent with mature neurons. The inset represents the magnification of framed area.
D-E. Mitotic activity within HESC-derived NP grafts as shown by dual staining for HNu and Ki67.
D. Mitotic profiles of NPs in grafts measured at 1.5, 3 and 6 months. Bars indicate group averages ± SEM. Variance in mitotic profiles between the three cohorts was significant (ANOVA, P = 0.014). Significant post hoc differences are indicated with brackets. * P < 0.05.
E. A representative area within a graft 3 months after transplantation, dually stained with HNu (red nuclear marker) and Ki67 (green nuclear marker). The three images illustrate the low rate of mitotic activity in HESC-derived grafts. The single double-stained nuclear profile (bottom) is indicated with an asterisk.
Abbreviations: Acb, nucleus accumbens; AOD, anterior olfactory nucleus, dorsal; AOV, anterior olfactory nucleus, ventral; Cx, cortex; cc, corpus callosum; GP, globus pallidus; Hy, hypothalamus; STh, subthalamic nucleus; Str, corpus striatum (neostriatum); th, thalamus; VP, ventral pallidum Scale bars: B-C, 100 μm (main panel), 10 μm (inset); E, 10 μm.

Astroglial differentiation of HESC-derived NPs in fornix (A-B), genu of corpus callosum (C) and internal capsule/globus pallidus (C-D). Sections were processed for ICC with GFAP (A) or human cytosol -specific SC121 (B, C-D) antibodies. Section in (A) was processed with immunofluorescence; sections in other panels were processed with immunoperoxidase.
A-B. In this case of spurious engraftment of human NPs into the fornix bundle, the vast majority of HNu (+) or SC121 (+) cells displayed astrocytic cytologies (both A and B) and GFAP expression (A). Insets in (A) represent a confocal microscopic analysis of the profile indicated by connecting lines; top inset is a traditional confocal appearance of the index profile with a subsequent virtual resectioning at the x- and y-axis; bottom inset is a compressed z-stack image of the index profile. Inset in (B) shows a low-power image of the fornix from which the main panel was derived.
C-D. In this case of a peripheral involvement of the callosal genu (C) or globus pallidus (D) in the graft, there was a predominant differentiation of human NPS into astrocytes. Insets in (C) and (D) are magnifications of framed areas in main panels.
Scale Bars: (A, C, D) 100 μm; (B) 10 μm.

Neural induction/differentiation of HESCs with noggin in adherent monolayer, from day 0 to P2.
A. Appearance of undifferentiated HESCs on MEFs at day 0 by phase contrast.
B. Induction/differentiation protocol used to generate NPs used in this study.
C. Day 0 (undifferentiated) HESCs express, by ICC, index pluripotency markers including Oct3/4, SSEA4, Tra1-60 and Tra1-81.
D. Oct3/4 immunoreactivity is substantially reduced in differentiating HESCs between days 9 (left) and 28 (right) in culture, evidence of shedding of HESC pluripotency.
Scale bars: 20 μm.

Immunocytochemical (ICC) and immunophenotypic profiling of HESC-derived NPs at the time of transplantation.
A-D. These single-epitope ICC preparations illustrate the expression of selective neural stem, neuronal and glial markers. Insets represent magnifications of areas shown with asterisks in the merged image. Although the vast majority of cells express the neural stem cell marker nestin (A), a tenth or less of them express the class III β-tubulin epitope TUJ1, a marker of immature neurons (B) or the glial (primarily astroglial) markers GFAP (C) and S100β (D).
E. These four-epitope ICC preparations serve to further characterize the phenotype of HESC-derived NPs at the time of transplantation. Epitope combinations are nestin-vimentin-A2B5-pax6 (top) and nestin-vimentin-musashi1-CD15 (bottom). Nestin colocalized extensively with vimentin (top, bottom) and musashi (bottom). Pax6, revealed with Alexa Fluor 750, was not present in these cells and is not shown here. A subpopulation of NPs expressed the early neuronal precursor marker CD15 (bottom), whereas very few expressed A2B5 (top). Asterisks indicate areas further enlarged in insets.
F. Immunophenotyping of HESC-NPs by flow cytometric analysis. HESC-derived NPs were dispersed into a uniform single-cell suspension and labeled with antibodies targeting CD133, CD29, CD15 and TnTx. Dead cells were counterstained with DAPI and the resulting vital phenotypes assayed by flow cytometry. Data are presented as two-parameter dot density plots in pseudocolor that reveal the relative abundance (percentiles) of different cell populations by using either electronically-gated regions as in (1) (Live Cells, Dead Cells, Debris) or quadrants as in (2-3). In (1), DAPI was used in conjunction with forward scatter to discriminate between live (DAPI-negative) and dead (DAPI-positive) cells. Data in (2-3) are based on live cells, which represent ∼80% of the preparation. In (2), the great majority of live cells are TnTx⁺ (∼79%, i.e. the sum of 48.21 and 30.73). CD15 labeling further subdivides these neuronal-restricted precursors into CD15⁺ (30.73%) and CD15^- (48.21%) subpopulations. CD15⁺TnTx^- NPs comprise 16.80% of total NPs. The CD133-versus-CD29 plot in (3) is gated on 4.26% of CD15^-TnTx^- cells (see panel F 2). Only 14.47% of these cells are CD29⁺, which amounts to ∼1% of total live cells (14.47 × 4.26/100). CD133⁺ cells represent less than 1% (0.75%) of the total population of live cells sorted.
Scale bars: 50 μm.

Neuronal differentiation of HESC-derived NPs in rat neostriatum. Photomicrographs represent cases of neuronal differentiation of HNu (+) cells by confocal microscopy from subjects prepared 3 months post-grafting.
A. Dual ICC for HNu and doublecortin shows many HNu (+) cells that are also immunoreactive for doublecortin (DCX).
B. Double ICC for HNu and TUJ1 demonstrates the abundance of double-stained cells in the grafts.
C. Bar graphs depicting neuronal cell fate choice in striatal grafts of HESC-derived NPs at 1.5, 3 and 6 months represented by percentages of DCX (left panel) and TUJ1 (right panel) -labeled HNu (+) cells. Bars indicate group averages ± SEM. Variance between the three cohorts was significant for both DCX (ANOVA, P = 0.0003) and TUJ1 (ANOVA, P = 0.0004). Significant post hoc differences are indicated with brackets. * P < 0.05; **P < 0.001.
Scale bars: 10 μm.

Evidence for some local and remote integration of differentiated human NPs in rat forebrain circuitry manifested by early (A) and late (B) pathway formation, remote synaptic configurations (C) and the formation of mature synapses as evidence by immuno-EM labeling for human synaptophysin and the human cytosolic marker SC121 (D-F).
A. Early projections from the 1.5-month graft visualized by doublecortin. Inset in (A), bottom, represents a confocal microscopic analysis, with a subsequent virtual resectioning at the x- and y-axis, of the framed area. B. Extensive formation of axon bundles by mature (6 month-old) differentiated grafts occurs along most established forebrain pathways including the striato-pallidal pencils and the anterior commissure (ac and sppen, top left), thalamic striae and the mamillothalamic tract (tstriae and mtt, top right), the rostral migratory stream (RMS, bottom left) and the genu-forceps minor of the corpus callosum (fm/cc, bottom right). In this panel, graft-derived axon bundles are visualized with SC121 ICC. Sections are counterstained with fast green.
C. Synaptophysin immunoreactivity in terminal fields in 6-month old grafts. These panels illustrate representative synaptic boutons in the olfactory bulb (OB) (internal plexiform and outer granule cell layer), layers I –II of frontal cortex (Cx), the globus pallidus (GP), and the islands of Calleja (IC). Insets represent magnifications of areas indicated with asterisks.
D-F. Electron microscopic images of synapses with human signature (D-E, human synaptophysin labeling; F, SC121 labeling) taken close to the striatal graft (D), in the nucleus accumbens (B) and in the glomerular layer of the olfactory bulb (C). Sections in (D) and (E) were prepared 3 months post-grafting; section in (F) was prepared 6 months post-grafting. Note postsynaptic densities (arrows) and synaptic vesicles in all labeled terminals.
Abbreviations: ac, anterior commissure; cc, corpus callosum; fm, forceps minor of corpus callosum; GR, graft; mtt, mammillothalamic tract; RMS, rostral migratory stream; sppen, striato-pallidal pencils; tstriae, thalamic striae.
Scale Bars: (A, B) 100 μm; (C) 10 μm.