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. 2009 Jul 14;17(10):1761–1770. doi: 10.1038/mt.2009.148

Conditions for Tumor-free and Dopamine Neuron–enriched Grafts After Transplanting Human ES Cell–derived Neural Precursor Cells

Ji-Yun Ko 1,2, Hyun-Seob Lee 1,2, Chang-Hwan Park 2,3, Hyun-Chul Koh 2,4, Yong-Sung Lee 1,2, Sang-Hun Lee 1,2
PMCID: PMC2835006  PMID: 19603007

Abstract

We have previously demonstrated derivation of neural precursor (NP) cells of a midbrain-type from human embryonic stem (hES) cells to yield an enriched population of dopamine (DA) neurons. These hES-derived NPs can be expanded in vitro through multiple passages without altering their DA neurogenic potential. Here, we studied two aspects of these hES-NP cells that are critical issues in cell therapeutic approaches for Parkinson's disease (PD): cell survival and tumorigenic potential. Neuroepithelial rosettes, a potentially tumorigenic structure, disappeared during hES-NP cell expansion in vitro. Although a minor population of cells positive for Oct3/4, a marker specific for undifferentiated hES cells, persisted in culture during hES-NP cell expansion, they could be completely eliminated by subculturing hES-NPs under differentiation-inducing conditions. Consistently, no tumors/teratomas are formed in rats grafted with multipassaged hES-NPs. However, extensively expanded hES-NP cells easily underwent cell death during differentiation in vitro and after transplantation in vivo. Transgenic expression of Bcl-XL and sonic hedgehog (SHH) completely overcame the cell survival problems without increasing tumor formation. These findings indicate that hES-NP cell expansion in conjunction with Bcl-XL+SHH transgene expression may provide a renewable and safe source of DA neurons for transplantation in PD.

Introduction

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by loss of dopamine (DA) neurons in the substantia nigra of the midbrain. Given the well-defined neuronal type and locus affected in PD, a cell transplantation approach offers great promise for the treatment of PD. Human fetal midbrain tissues have been clinically used as a source of DA neurons for transplantation in PD patients. However, this supply is limited and difficult to develop as a routine systematized source. Furthermore, the clinical benefits of fetal nigral transplantation in PD patients are controversial.1,2 Stem cells with self-renewal capacity and multidevelopmental plasticity are regarded as an alternative and attractive source of cells for replacement strategies, not only because they are capable of yielding consistently functional DA neurons on a large scale, but also for their potential to provide clear behavioral recovery in PD patients upon transplantation.3

Human embryonic stem (hES) cells are primitive, undifferentiated cells derived from the inner cell mass of preimplanted human blastocysts. These cells can be efficiently guided to differentiate toward a neuronal lineage, producing enriched populations of cells exhibiting in vitro functions typical of presynaptic DA neurons.4,5,6 Although these attributes suggest that hES-derived DA cells may be a desirable source of cells for cell replacement strategies, the prospects of using hES cells for PD cell therapy remain uncertain given inconsistent outcomes reported in PD model rats, including tumor formation, and limited survival and function of grafted hES-derived DA neurons. In most reports, relatively few (<500 cells per animal) tyrosine hydroxylase (TH)-positive DA cells survive, with minimal-to-moderate (<50%) behavioral restorations in PD rats transplanted with hES-derived DA cells.6,7,8,9,10 One exception was a report by Roy et al.11 that demonstrated the presence of an exceptionally large number of hES-derived TH+ cells in the striatum of transplanted rats and major behavioral improvements. However, these promising results were largely negated by the presence of large tumorigenic cores composed of proliferating cells, which were observed in the centers of the grafts. A recent study12 has reported that up to 1,300 TH+ cells per graft survived for a relatively long period (5 months) after transplantation without tumor formation. However, the ability to obtain large numbers of TH+ DA neurons without tumor formation in hES cell–based PD transplantation remains far from satisfactory; further extensive systematic evaluations are clearly required.

The diverse outcomes of PD transplantation models are mainly attributable to differences in the protocols applied in hES differentiation and donor cell preparation. These differences, in turn, affect cell viability, cell composition, cellular types, and developmental stages of the donor cells, suggesting the need for a standardized methodology in future systematic analyses. Most hES differentiation protocols currently in use inevitably generate a mixture of cells at different developmental stages and include cells of unwanted tissue types. In addition, hES cell differentiation in vitro can be highly variable depending on hES cell lines, passage numbers, and culture conditions. In an effort to solve these problems, we have recently reported a procedure that induces hES cell differentiation toward a highly homogeneous population of neural precursor (NP) cells that could give rise to enriched numbers of DA neurons upon terminal differentiation.5,6 Furthermore, the proliferative and DA neurogenic potential of the hES cell–derived NP (hES-NP) cells are maintained after long-term NP cell expansion and multiple freeze–thaw cycles.6,13 These results suggest that the NP cells derived by our hES cell protocol could provide a continuous, stable, and on-demand experimental system that can be used to optimize cell therapeutic conditions based on hES cells. In the present study, we assessed cell survival and tumorigenic potential of our hES-NP cells in order to provide improved donor cell preparation for generating tumor-free graft enriched with DA neurons after transplantation; especially we pursued whether these two critical aspects of the hES-NP cells could be altered by extended NP cell expansion with multiple passages and in in vitro conditions that mimic the in vivo environment of the transplant by eliminating trophic factors supplemented in culture for cell proliferation and differentiation.

Results

Proportions of potentially tumorigenic cells in hES-NP cell cultures

Our protocol for derivation of hES-NP cells includes (i) neural induction on the MS5 stromal cell feeder, (ii) ventral midbrain patterning by treatment with sonic hedgehog (SHH) + fibroblast growth factor 8 (ref. 14), (iii) successive subculturing of completely dissociated cell preparations, and (iv) expansion of NP cells with basic fibroblast growth factor (bFGF). The yield is a highly homogeneous (>95% nestin-positive cells) population of midbrain-type NP cells that terminally differentiate into DA neurons co-expressing the midbrain DA neuron markers, Engrailed-1, Nurr1 (NR4A2), and G protein–gated inward rectifier K+ channel (Girk2) (Supplementary Figure S1).5,6 The population remains homogeneous and DA neuron enriched through multiple cycles of passage and expansion6,13 as required of a stable and renewable cell source for transplantation. For successful therapy, however, donor cells must be free of tumorigenicity and able to survive in vivo. To test the first requirement, we determined the proportions of undifferentiated hES cells and the cellular composition of neural rosettes (neuroepithelial cells), both reported to generate tumors (e.g., teratomas), in hES cell–derived transplants.8,11,15,16 The hES-NP single cell dissociates, when plated for subculture, reformed numerous rosette structures expressing the NP cell marker nestin and the proliferating cell marker Ki67 during the first three passages (Figure 1a left, b). However, numbers of these primitive neuroepithelial structures were decreased during 2–4 passages of NP cell expansion [7.9 ± 0.4/cm2 at NP passage 2 (P2) versus 5.4 ± 0.1/cm2 at P4, P < 0.01 by analysis of variance with post hoc Tukey t-tests; F = 203.859 (2, 6 degrees of freedom (d.f.)); obtained from nine dishes (6 cm) of each passage in three independent experiments], and completely depleted after six passages (Figure 1c), indicating that the use of late NP cells for transplantation may reduce tumor risk. Appearance of rosette structures in early passages and elimination by several passages were invariable regardless of hES cell line (HSF6, H9) and hES-NP cell stock. By contrast, proportions of undifferentiated hES cells detected by Oct3/4 immunostaining were highly variable (0–5% out of total cells) in hES-NP cells generated by different rounds of hES cell differentiation. Thus, experiments were followed using hES-NP cell stocks containing high percentages of Oct3/4+ cells. A proportion of cells (4.8 ± 0.1%, n = 3, 90 microscopic fields from nine cultured coverslips of three independent experiments) expressed the undifferentiated hES cell marker, Oct3/4, when determined 4 days after bFGF expansion of NP passage 1 (NP-P1: Figure 1d,e). These Oct3/4+ cells gradually decreased over passages [for example, 2.4 ± 0.2% at P10, significantly different from P1 at P < 0.01, F = 46.649 (3, 8 d.f.)], but did not completely disappear even after 10 cycles of complete dissociation and replating in the cell passage procedure.

Figure 1.

Figure 1

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Proportion of potentially tumorigenic cells in hES-NP cell cultures. (a–e) Neural rosette formation (a–c) and proportions of Oct3/4+ undifferentiated hES cells (d,e) in expanded hES-NP cultures at sequential cell passages. The hES-NP cells were passaged every 7 days in the presence of basic fibroblast growth factor (bFGF) by dissociation into single cells and plating on poly-L-ornithine/fibronectin-coated dishes. Images in (a) are the representative phase contrast microscopic views of hES-NP cultures at NP passage 2 (P2, left) and P6 (right); (b) immunofluorescent view of nestin+/Ki67+ neural rosette structures in hES-NP at P2; (c) graph of numbers of rosettes counted; (d) representative images of Oct3/4+ cells in hES-NP cultures at P1, P5, P8, and P10; (e) the percent of total cells that were Oct3/4+. *Significantly different from NP passage 2 (or 1) at P < 0.01. (f,g) Proportion of Oct3/4+ cells among directly differentiating hES-NP cells. NP-derived cells from P5 were dissociated, plated at 0.8 × 105 and 7.5 × 105 cells/well (a cell density identical to that of donor cell solutions used for transplantation; see Materials and Methods), and directly differentiated in the absence of bFGF for 1 (Dif. 1), 4, and 6 days. Note that there are no Oct3/4+ cells after culturing under bFGF-free conditions for 4 or 6 days. Bar = 40 µm. DAPI, 4′,6 diamidino-2-phenylindole; hES, human embryonic stem; NP, neural precursor.

Although cells were initially plated in the form of single cell dissociates, virtually all Oct3/4+ cells assembled into cell clusters. The presence of such clusters suggests that dissociated Oct3/4+ cells have the potential to survive and proliferate. Given that bFGF is indispensable for the maintenance and proliferation of undifferentiated hES cells, this factor could be considered most important for the survival/proliferation of Oct3/4+ cells. Thus, to more appropriately predict the possibility of teratomas after hES-NP cell transplantation, we re-evaluated the proportion of Oct3/4+ cells after culturing in the absence of bFGF, a condition that mimics the in vivo environment of the transplant where high levels of bFGF are unlikely. One day after plating hES-NPs in bFGF-free media, Oct3/4+ cells were detected in the form of dissociated single cells (percent Oct3/4+ cells: 2.1 ± 0.3% at P2, 1.8 ± 0.3% at P5, and 1.4 ± 0.2% at P10, n = 3, 90 microscopic fields from nine coverslips of three independent cultures). Surprisingly, after culturing for three more days in the absence of bFGF, Oct3/4+ cells completely disappeared and did not reappear during the rest of the culture period (Figure 1f,g). The disappearance of Oct3/4+ cells followed the same pattern regardless of NP passage number (data not shown), and was also evident in bFGF-free cultures plated at the extremely high cell concentration (7.5 × 105 cells/well in 24-well plates; Figure 1f,g); the number of cells is identical to that used for transplantation (see Materials and Methods).

Apoptosis of hES-NP cells after multiple passages in the absence of trophic factors

In most previous studies of hES-DA differentiation,4,5,6,12,13 terminal differentiation of hES-NP cells into DA neurons was induced in the presence of differentiation and survival factors such as brain-derived neurotrophic factor (BDNF), glial cell line–derived neurotrophic factor (GDNF), and cyclic adenosine monophosphate (cAMP). In the continued presence of these cytokines, cells survived for >15 days of in vitro differentiation with no change in viability or apoptotic indexes with passage number (data not shown).6 However, high levels of these trophic factor supports are unlikely in the transplanted brain in vivo. Thus, an appropriate prediction of hES-NP cell survival and differentiation after transplantation needs to be evaluated in the absence of these trophic factors. Without the supplemental trophic factors, cell viability fell precipitously after more than five passages (late NP), and the cultures failed to survive for longer than 4–6 days of differentiation. Consistently, the indexes of apoptosis [estimated by the number of cells with apoptotic nuclei and cells positive for activated (cleaved) caspase-3 (Figure 2)] gradually increased with passage number. Three days after bFGF withdrawal, for example, the proportions of cleaved caspase-3+ cells in cultures of P1, P5, and P10 were 3.6 ± 0.6%, 7.0 ± 0.4%, and 35.0 ± 0.9%, respectively (for each value, n = 3, 90 microscopic fields from nine coverslips of three independent cultures). The proportions of apoptotic nuclei also increased significantly (P1: 4.2 ± 0.5%, P5: 9.1 ± 0.4%, and P10: 41.5 ± 1.2%, Figure 2b). This trophic factor dependence would prohibit the use of the hES-NP cells for transplantation therapy, and this finding guided our subsequent investigation.

Figure 2.

Figure 2

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Apoptosis of hES-NP cells upon differentiation increases with passage number. At different passage numbers, hES-NP cells were cultured in the presence of basic fibroblast growth factor (bFGF) for 5 days and induced to differentiate by withdrawal of bFGF. At day 3 of differentiation, apoptosis was measured by immunocytochemical analysis for activated (cleaved) caspase-3. Images in (a) are representative for activated caspase-3+ cells in cultures with NP passages 1–10; (b) the percent of cells immunoreactive to activated caspase-3 (green bar) and cells with apoptotic (condensed or fragmented) nuclei (red bar), respectively. *Significantly different from NP passage 1 [P < 0.01, F = 629.417 (9, 20 d.f.) for caspase-3+ cells and F = 1086.768 (9, 20 d.f.) for apoptotic nuclei counting, n = 3, 90 microscopic fields from nine coverslips, of three independent cultures]. Bar = 40 µm. DAPI, 4′,6 diamidino-2-phenylindole; hES, human embryonic stem; NP, neural precursor.

Effects of BDNF, GDNF, and cAMP on cell survival and DA neuron yields from hES-NP cells

We next examined the contribution of each trophic factor to the survival of differentiating hES-NP cells. Individual treatments of BDNF, GDNF, and cAMP slightly decreased apoptosis (Figure 3a,b). By contrast, the triple combination of BDNF+ GDNF+cAMP dramatically improved cell survival. At day 3 differentiation in passage 8 (P8), the proportions of total 4′,6 diamidino- 2-phenylindole-positive cells that were cleaved caspase-3+ were 11.5 ± 0.7% with the triple treatment and 29.2 ± 1.1% in the control [n = 4, 240 microscopic fields from 12 coverslips, of four independent cultures; P < 0.01, F = 34.269 (7, 24 d.f.)]; cells with apoptotic nuclei were 11.4 ± 0.6% and 28.0 ± 1.1% of the total in treated and control cultures, respectively [P < 0.01, F = 30.673 (7, 24 d.f.)].

Figure 3.

Figure 3

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Effects of BDNF, GDNF, and cAMP treatment on survival and differentiation of hES-NP cells with multiple passages. (a,b) Cell survival and (c,d) dopamine neuron yields were estimated at P8 by the percent of total (DAPI+) cells with cleaved caspase-3+/apoptotic nuclei and positive TH reaction (TH+) 3 days after differentiation in the absence or presence of the cytokines. The percent of early hES-NP cells (P2) that were TH+ was measured (e) over 15 days of in vitro differentiation in the presence (red dots) or absence (blue dots) of BDNF+GDNF+cAMP. *P < 0.01 from the respective control values. Bar = 40 µm. BDNF, brain-derived neurotrophic factor; cAMP, cyclic adenosine monophosphate; DAPI, 4′,6 diamidino-2-phenylindole; GDNF, glial cell line–derived neurotrophic factor; hES, human embryonic stem; NP, neural precursor; TH, tyrosine hydroxylase.

During 15 days of hES-NP cell differentiation in vitro, the numbers of TH+ DA neurons increased gradually. In the absence of the trophic factors, DA neuron differentiation slowed and decreased (Figure 3e). Surprisingly, but consistent with previous reports,17,18,19,20 TH+ cell numbers in the cultures treated with BDNF or GDNF did not differ significantly from the untreated control. At day 6 differentiation, the proportions of TH+ cells were 2.1 ± 0.3% in control cultures, 2.6 ± 0.3% in BDNF (P = 0.92), and 2.7 ± 0.2% in GDNF (P = 0.83). By contrast, cAMP treatment greatly enhanced TH+ cell yields [7.3 ± 1.0%; Figure 3c,d, n = 4, 80 microscopic fields from eight coverslips, of four independent cultures; P < 0.01, F = 191.619 (7, 24 d.f.)]. Further improvement of TH+ cell yields was shown with combined treatments of cAMP+BDNF (11.9 ± 0.4%) and cAMP+BDNF+GDNF (13.0 ± 0.6%). In this experimental context, the triple combination of factors provided optimal support for hES-NP cell survival and differentiation in vitro.

Bcl-XL and SHH improve survival of differentiating hES-derived NP cells

The low viability of cytokine-deprived late hES-NP cells severely limits their potential use as a cell source for transplantation. We therefore explored ways to sustain their survival through manipulation of cell survival–related genes. The Bcl-XL protein, a member of the antiapoptotic Bcl-2 family, has been shown to prevent cell death in various neuronal populations.21 In NPs22 and postmitotic neurons, especially midbrain DA neurons,23 SHH has also been shown to mediate survival. We tested whether transgenic expression of these survival factors could augment the viability of late NP cells. In contrast to the low efficiency of retroviral gene transfer in undifferentiated hES cells (data not shown), a retroviral construct efficiently expressed the transduced genes in hES-derived NP cells: 2 days after infection with a retroviral construct expressing green fluorescent protein, >40% of total cells were green fluorescent protein–positive, and the expression was sustained for at least 15 days after infection (Figure 4a,b). Similarly, expression of Bcl-XL and SHH exogenes, estimated by reverse transcription–PCR analyses, was robustly induced in hES-NP cells by retroviral gene transfer and stably maintained during the culture period (Figure 4b). Estimates of cell death and apoptosis by lactate dehydrogenase assay (Figure 4e), staining for propidium iodide (Figure 4c,f) and cleaved caspase-3 (Figure 4d,g) showed that coexpression of Bcl-XL and SHH enhanced cell survival additively or synergistically in hES-NP cells at P10. Proportions of cells positive for cleaved caspase-3, for example, were 42.2 ± 1.3% (LacZ control) 33.0 ± 0.5% (Bcl-XL), 27.0 ± 2.0% (SHH), and 6.5 ± 0.8% (Bcl-XL+SHH). Most importantly, late hES-NP cells (P5–P10) expressing Bcl-XL+SHH (but not Bcl-XL or SHH alone) could survive for >15 days of in vitro differentiation in the absence of cAMP+BDNF+GDNF. These genetic manipulations, however, did not significantly alter yields of TH+ DA neurons (data not shown).

Figure 4.

Figure 4

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Transgenic expression of Bcl-XL and SHH in late hES-NP cells prevents cell death and apoptosis. The hES-NP cells at P10 were transduced with retroviruses expressing Bcl-XL and/or SHH as described in Materials and Methods. Differentiation was induced the day following transduction by withdrawal of bFGF, and cell death or apoptosis was analyzed 3 days later. (a,b) Efficiency and pattern of exogene expression were estimated in cultures transduced with viruses carrying GFP, Bcl-XL, and SHH genes during 15 days of in vitro differentiation. Shown in (a) are images of GFP-immunoreactive cells in the cultures at different days after inducing differentiation; (b) Reverse transcription–PCR analyses of exogenous Bcl-XL and SHH mRNA expression during the course of in vitro differentiation. The PCR conditions will be provided upon request. (c–g) Indexes of cell survival/death in cultures transduced with LacZ (control), Bcl-XL (B), SHH (H), or Bcl-XL+SHH. Representative images for (c) PI+ cells and (d) activated caspase-3+ cells in the indicated transduced cultures (Bar = 40 µm); (e) LDH release relative to the respective LacZ control; (f) percent of cells immunoreactive for PI and activated caspase-3; and (g) percent of cells with apoptotic nuclei. Significantly different from *LacZ-transduced control, **Bcl-XL-transduced, and ***SHH-transduced at P < 0.01. The statistical analyses were made by ANOVA with post hoc Tukey comparisons [n = 12 cultures from three independent experiments, F = 49.330 (3, 48 d.f.) for LDH assays; n = 3, 60 microscopic fields from six cultured coverslips of three independent cultures, F = 76.371 (3, 8 d.f.) for PI-stained cells; n = 3, 120 microscopic fields from six coverslips of three independent cultures, F = 143.522 (3, 8 d.f.) for cleaved caspase-3+ cells and F = 142.415 (3, 8 d.f.) for cells with apoptotic nuclei]. DAPI, 4′,6 diamidino-2-phenylindole; G3PDH, glyceraldehyde-3- phosphate dehydrogenase; GFP, green fluorescent protein; hES, human embryonic stem; LDH, lactate dehydrogenase; NP, neural precursor; PI, propidium iodide; SHH, sonic hedgehog.

Enhanced survival of Bcl-XL+SHH-transduced hES-NP cells in PD rats

In the final set of experiments, we explored the in vivo survival, differentiation, and function of hES-NP cells at early and late passages, and the effects of Bcl-XL+SHH expression in these cells when they were transplanted to the striatum of the rat PD model. The PD rats were randomly assigned into five groups and grafted with naive early hES-NP cells (NP-P2 and NP-P3, n = 9, early NP), early NP cells transduced with Bcl-XL+SHH (n = 16, B+H early NP), late NP cells (P9 and P10, n = 7, late NP), and Bcl-XL+SHH-transduced late NP cells (n = 16, B+H late NP). An additional group was sham operated [phosphate-buffered saline (PBS)-injected, n = 5]. The hES-NP cells were injected directly into the rat striatum.

Naive, nontransduced early NP cells survived in the host striatum and formed masses of grafts along the needle tracts (Figure 5a,e). Average graft volume in these animals was 1.5 ± 0.5 mm3 at 8 weeks post-transplantation. The total number of viable donor cells per graft labeled by immunostaining of human nuclear antigen (HN+ cells) was 483,814 ± 128,923 cells (Figure 5f,j), resulting in a density of 198,167 ± 38,751 HN+ cells per mm3 in the graft. In contrast, a few HN+ cells were detected occasionally in the striatum of rats grafted with late NP cells (Figure 5h,j). Transplants of late NP cells did not form graft masses at all (Figure 5c,e,h,j). The survival indexes in PD rats grafted with B+H early NP cells did not differ significantly from those of early NP cells (Figure 5b,e,g,j). By contrast, B+H transduction in late NP cells strikingly enhanced survival indexes in vivo (Figure 5d,e,i,j). Graft volumes (1.4 ± 0.3 mm3) and HN+ cells (492,596 ± 243,097 cells) in animals grafted with B+H late NP cells were similar to those of animals grafted with early NP cells. These results show that B+H manipulation of late NP donor cells can completely prevent loss of viability post-transplantation.

Figure 5.

Figure 5

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Effect of Bcl-XL+SHH on in vivo survival of hES-NP cells. Cultures of hES-NP cells were prepared and transplanted to Parkinson's diseased (PD) rats as described in Materials and Methods. The animals were killed 8 weeks after transplantation. Presented here are (a–d) representative light microscopic views of grafts in the striatum of PD rats transplanted with (a) naive early NP (early NP), (b) early NP transduced with Bcl-XL+SHH (B+H early NP), (c) late NP nontransduced (late NP), and (d) B+H transduced (B+H late NP) (Bar = 200 µm), (e) the graft volume for each animal group, (f–i) immunofluorescent images for HN+ grafts (Bar = 40 µm); (j) graph showing total numbers of HN+ cells in the grafts of the animals, (k–m) representative images for (k) cells positive for nestin/4′,6 diamidino-2-phenylindole (DAPI), (l) PCNA/DAPI, and (m) pHH3/DAPI in the rats transplanted with B+H early NP, and (n) representative microscopic image of hematoxylin and eosin–stained graft of early NP B+H. Dashed lines mark the borders of the grafts (Bar = 40 µm). hES, human embryonic stem; NP, neural precursor; PCNA, proliferating-cell nuclear antigen; pHH3, phospho-Histone H3; SHH, sonic hedgehog.

The cells within grafts were morphologically uniform with no evidence of neoplastic change or tumor formation in hematoxylin and eosin–stained tissues regardless of NP cell passage number and B+H transduction (Figure 5n and data not shown). Although neural rosette structures did form in early NP cell cultures, the cells grafted did not generate neural rosettes by 8 weeks after transplantation. Subpopulations, however, were positive for the NP cell marker nestin and the proliferating cell markers proliferating-cell nuclear antigen and phospho-Histone H3 in all the grafts generated by early and late NP cells (Figure 5k–m). Transduction with B+H did not significantly affect those immunoreactive cell populations or the shape of the resulting graft.

Optimal immunohistochemical detection of TH+ DA neurons in grafts of hES-NP cells

Most previous studies of hES cell–based transplantation in PD reported unexpectedly low yields of TH+ DA neurons in the grafts.5,7,8,9,10 These low yields of hES cell–derived DA neurons in vivo contrast clearly with findings of viable DA neurons in abundance in brain grafts of mouse embryonic stem–derived NP cells.24,25,26 Human DA neurons are more vulnerable to oxygen free radicals produced by TH enzyme activity than rodent DA neurons, and may therefore maintain only low levels of TH expression.27,28 For this reason, the low DA neuron numbers in the transplants of hES-derived cells may be due to inefficient detection of TH antigenic determinants. We thus varied the conditions of TH immunofluorescent staining in an effort to optimize results. Among the antibodies tested, TH antibody from Pel-Freez (see Materials and Methods) showed the highest sensitivity to hES-derived DA neurons in the grafts, giving the strongest positive signals under all conditions tested (data not shown). Inclusion of biotin–streptavidin amplification step,29 modifications in the composition of blocking solution, fixative, and incubation time with the primary TH antibody had no apparent effect on the staining. However, antigen retrieval by sodium dodecyl sulfate (SDS) treatment (see Materials and Methods) greatly improved the TH sensitivity of immunohistochemical detection of hES-DA neurons in vivo (Figure 6). Using the SDS method, we detected approximately ninefold more TH+ cells, with stronger intensity of the positive signals, than without the treatment [131 ± 34 cells/graft without and 1,262 ± 207 cells/graft with SDS treatment; Figure 6d, n = 3 grafts for each value; P < 0.01, F = 21.195 (2, 51 d.f.)]. The boiling method for antigen retrieval (see Materials and Methods) also enhanced TH+ signal intensity and the number of TH+ cells detected, but did not increase detection sensitivity to the degree that the SDS treatment did. The increased detection sensitivity with SDS treatment was further confirmed by comparing TH+ cell numbers in identical sections (Figure 6e–g). We therefore used the SDS antigen retrieval method for all TH staining of sectioned tissues in this study.

Figure 6.

Figure 6

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Antigen retrieval improves the sensitivity of TH immunohistochemical detection for dopamine neurons in hES-NP–derived grafts. (a–d) Comparison of TH+ cell numbers detected without (a) and with antigen retrieval using the boiling method (b) or SDS treatment (c) in adjacent sections of the same graft. Eight weeks after transplantation, brain slices from three rats grafted with B+H late NP cells were prepared. Shown in (a–c) are representative images for TH+ cells in the grafts obtained from adjacent sections with the different methods. Inset: high-powered view of the boxed area. Graph (d) depicts the average TH+ cell numbers of three grafts calculated as described in Materials and Methods. Significantly different from *untreated and **boiling method at P < 0.01, n = 3. (e–g) Comparison of TH+ cell detection sensitivity between control (no treatment) and SDS-treated methods in identical sections. Ten sections (each derived from different animals) were initially subjected to TH immunostaining without antigen retrieval, and TH-stained cells per section counted. Subsequently, identical sections were carefully washed and stained with SDS, and TH+ cells counted. Shown in (e,f), respectively, are images for TH+ cells stained with untreated and SDS-treated methods in an identical section. Arrows indicate an identical TH+ cell in the images. Graph (g) represents the relative values of TH+ cells to those of control untreated cells. *Statistical comparisons were made between TH+ cell numbers detected with the two methods in each identical section using the paired t-test (n = 10, t = −2.716, P = 0.024). Bar = 20 µm. DAPI, 4′,6 diamidino-2-phenylindole; hES, human embryonic stem; NP, neural precursor; SDS, sodium dodecyl sulfate; TH, tyrosine hydroxylase.

TH+ DA neuron yield and behavioral assessment of PD rats grafted with hES-NP cells

In the animals with grafts of early NP cells, grafts showed an average of 976 ± 366 TH+ cells (Figure 7a,i), approximately five times the number we previously detected in hES-NP cell transplants without using the SDS treatment.6 Transplanting early NP cells transduced with Bcl-XL+SHH produced a similar yield of TH+ cells (1,140 ± 276 cells; Figure 7b,i). In rats that received nontransduced late NP cells, we detected neither HN+ graft formation (Figure 5h,j) nor TH+ graft formation (Figure 7c,i). However, the numbers of TH+ cells in rats grafted with B+H late NP cells were similar to the numbers in grafts of early NP cells (1,056 ± 342 cells, Figure 7d,i). The TH+ cells in grafts were morphologically mature, with extensive neurite outgrowth (insets of Figures 6c and 7e–h) and abundant expression of the midbrain DA neuronal markers was detected in hES-NP–derived grafts (Figure 7j).

Figure 7.

Figure 7

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In vivo analyses of hES-NP cells transplanted to Parkinson's diseased (PD) rats. hES-NP cells at different NP passage numbers with or without B+H transduction were prepared and injected into PD rats as described. (a–i) Dopamine (DA) neuron yields from hES-NP cells transplanted into PD rats. Immunostaining for TH was performed using the sodium dodecyl sulfate (SDS) antigen retrieval method as described. For TH/HN double immunostaining (a–d), the tissue sections were initially stained with HN without SDS treatment, followed by TH staining with SDS treatment. The TH and TH/HN images were obtained by stacking z-series through the thickness of the section (35 µm). Shown in (a–h) are confocal microscopic images of TH+/HN+ (a–d) and TH+ (e–h) cells in the striatum of rats grafted with the indicated hES-NP cells. (h) An image from an isolated TH-stained DA cell with well-defined neurite outgrowths obtained from a graft of hES-NP at P2. Bar = 40 µm. Graph (i) depicts total numbers of TH+ cells in the grafts of the animals. (j) Midbrain DA neuron marker expression in hES-NP cell grafts. After transplantation with B+H-transduced NP-P9, RNA samples were prepared from ipsilateral graft (G) and contralateral host striatum (H). Reverse transcription–PCR analysis of markers specific for general (TH, VMAT2) and midbrain-type (Nurr1, En1, Lmx1b, Girk2, and Ptx3) DA neurons. (k) Amphetamine-induced rotation test. For the sham control, five animals were injected with phosphate-buffered saline (PBS) by the same schedule as the cell-grafted animals. Each value depicts mean ± SEM of % changes in rotation scores as compared to pretransplantation values. Statistical comparisons were made by analysis of variance, followed by the Scheffé test. *P < 0.05 from PBS-injected group, n = 9 (early NP), n = 16 (B+H early NP), n = 7 (late NP), and n = 16 (B+H late NP). DAPI, 4′,6 diamidino-2-phenylindole; hES, human embryonic stem; NP, neural precursor; TH, tyrosine hydroxylase.

Consistent with the TH+ cell numbers and morphological maturity in the transplants, animals grafted with early NP cells showed significant reductions in amphetamine-induced rotation scores in 8 weeks after transplantation (Figure 7k). Compared with pretransplantation values, the rotation scores at 8 weeks were 47 ± 13% [P < 0.05, F = 5.665 (4, 40 d.f.)] for early NP transplants and 45 ± 10% [P < 0.05, F = 6.288 (4, 75 d.f.)] for those with B+H early NP cells. None of the animals grafted with nontransduced late NP cells showed a reduction in the rotation score. Average amphetamine-induced rotations at 8 weeks increased relative to preimplantation values in these animals (111 ± 6%). By contrast, 8 of 16 rats grafted with B+H–transduced late NP cells showed reductions in rotation scores of >50%. Average rotation score in animals with B+H late NP transplants was 50 ± 9% [P < 0.05, F = 5.842 (4, 75 d.f.)] of the preimplantation value at 8 weeks after transplantation. The rotation results were also expressed as the numbers of turns per hour in Supplementary Figure S2. These data indicate that manipulation of Bcl-XL and SHH genes together may overcome the loss of viability and functional activity of late hES-NP cells in transplants to PD rats.

Discussion

Previous works6,13 established that hES-derived NP cells maintain their proliferative capacity and dopaminergic phenotype through multiple cell passages; through expansion, this cell type can generate the large, stable, homogeneous populations required for transplantation therapy of PD. We have now shown that extended passage of these NP cells depletes neuroepithelial rosette structures, and may thereby contribute to a decrease in their tumorigenic potential after transplantation. The extended cell expansion period with multiple passages also resulted in a significant reduction in the percentage of Oct3/4+ undifferentiated hES cells during expansion, although the subculture procedures could not completely eliminate undifferentiated cells. These findings suggest a substantial possibility of teratoma formation after hES-NP cell transplantation. However, consistent with previous reports,7,12,30 none of the animals grafted with our hES-NP cells showed overt tumor formation for at least 2 months after transplantation. A plausible explanation for the absence of teratoma formation after transplantation of hES-NP cells is our interesting observation that the survival of Oct3/4+ cells in hES-NP cell cultures was absolutely dependent on bFGF supplementation; undifferentiated cells were completely eliminated by simply culturing for several days in bFGF-free media. Because the transplanted brains surely do not provide a bFGF-enriched environment, any undifferentiated cells that were injected should not survive in the host brains. This finding suggests that the possibility of teratoma formation after transplantation of hES-NP cells might be lower than expected based on the number of Oct3/4+ cells present in the hES-NP donor cell solution. However, at the clinical level, donor cell preparation requires steps capable of completely eliminating undifferentiated cells. As indicated, preculturing hES-NP cells in bFGF-free media for several days before transplantation might be a simple practical way to generate donor NP cells for clinical applications.

Cell viability steadily declined with passage, however, and virtually none of the late NP cells survived in a graft (Figures 2 and 5h,j). We then showed that transgenic expression of Bcl-XL and SHH in hES-NP cells additively enhances their survival and prevents depletion of late NP cells in brain transplants in vivo (Figures 4 and 5i,j). In addition to its antiapoptotic role, Bcl-XL participates at various stages of neuronal differentiation, including cell fate determination,31,32 maturation, and acquisition of functional properties in cultures of rodent neurons.33,34 In mouse-derived embryonic stem cells, the overexpression of Bcl-XL enhances the generation and maturation of DA neurons and the development of dopaminergic activity.26 SHH is a signaling factor that emanates from the floor plate of the developing brain. This secreted molecule continues to be expressed in the adult brain, where it plays a role in promoting the survival35 and proliferation36 of NP cells. The SHH-mediated ventralizing effect in the developing brain is associated with induction of DA neuronal fate determination in early embryonic midbrain precursors.14,37 Consistent with this observation, SHH cytokine treatment in mouse embryonic stem–derived NP cells has been shown to enhance the yield of DA neurons in culture.38 By contrast, neither Bcl-XL nor SHH showed similar effects on neuronal or DA neuronal differentiation in hES-NP cultures. Further study may reveal mechanisms for the species-dependent differences of the cytokine effects.

Most investigators report detection of no or very few (0–400) TH+ cells in vivo after transplanting hES cell derivatives.5,6,7,8,9,10 Similarly, transplantation of MESC2.10 cells from a human mesencephalic cell line did not generate TH+ cells in the striatum of PD rats even though the cells exhibit features of DA neurons in vitro.39 This contrasts sharply with the enrichment of TH+ DA neurons in the brains of PD rats receiving mouse embryonic stem-derived NP cells24,25,26 or rat embryonic midbrain cells directly.19 The DA neuron yields in transplants may therefore represent an important distinction between human DA neurons and those of rodents. There are several possible causes for the poor DA neuron yields in the grafts generated by human cells. Similar to our findings here (Figure 3), Paul et al. found that TH expression in human DA cells disappears in a medium lacking differentiation factors (i.e., cAMP and neurotrophins),39 but could be restored by repletion of those factors, indicating unstable TH expression of human DA cells depending on the trophic factors. Thus, the trophic factor dependency of human cells in their TH expression may be a prime cause for the relatively few TH+ cells in the human neuronal transplants because enrichment of these trophic supports cannot be expected in the host environment. Second, low TH+ cells of human cell transplants may stem from suboptimal conditions for immunohistochemical detection of TH in previous measurements, which would underestimate the numbers of human DA neurons in the grafts. We base this assertion on our experience and communications with others, having shown distinct differences between rodent and human cells with respect to antigenic properties and detection sensitivities for several DA neuronal markers. Detection sensitivity may be especially important because these cells are extremely sensitive to oxidative injury and may normally maintain low levels of TH expression to minimize the oxidative stress imposed by TH activity.27,28 As we show here, antigen retrieval with SDS treatment can significantly improve immunohistochemical sensitivity to TH determinants in human DA cells in vivo.

Given the restricted access to human tissues and cells, most investigators of midbrain dopaminergic function have used rodent models and DA cell lines. But although human and rodent midbrain DA neurons share many properties, they differ sharply in important ways, including midbrain marker gene expression and response to mitogens, differentiation factors, and conditions in their tissue environment. The need for neurological therapies grows, and with it, the need for experimental systems based on human cells. In this regard, our hES-NP cells provide a stable, renewable population for studies of human dopaminergic function and optimization of cell therapy for PD.

Materials and Methods

Culture and differentiation of hES cells. NP cells with a midbrain phenotype were generated from hES cells (HSF-6, established at University of California, San Francisco, CA, passages 53–86) as described previously.5,6 Briefly, hES cells were neurally induced on a feeder layer of MS5 stromal cells, and the cocultures were subcultured several times until >90% of hES cell colonies contained neuroepithelial structures with neural rosettes (hES-NP colonies). For ventral midbrain patterning of hES-NP colonies, the final 1–2 rounds of subcultures were maintained on MS5 feeder cells stably overexpressing SHH5 in the presence of fibroblast growth factor 8 (100 ng/ml; R&D Systems, Minneapolis, MN). At the end of the coculture, NP colonies were harvested using collagenase (collagenase type IV, 1 mg/ml; Invitrogen, Grand Island, NY), gently triturated by pipetting into clusters of 50–500 cells in ITS (insulin/transferrin/selenium) plus ascorbic acid (AA, 0.2 mmol/l; Sigma, St Louis, MO), supplemented with bFGF (20 ng/ml; R&D Systems), and replated onto poly-L-ornithine (15 µg/ml; Sigma)/fibronectin (1 µg/ml; Sigma)-coated dishes. After culture for 7–9 days, hES-derived NP clusters were dissociated into single cells by exposure to Ca2+- and Mg2+-free Hank's balanced salt solution for 1 hour at 37 °C. These were then replated at 2.5 × 105 cells/cm2 in ITS + AA + bFGF (NP-P1). Cells were passaged every 7 days in medium supplemented with bFGF. For phenotypic determinations, hES-NP cells were plated on poly-L-ornithine/fibronectin-coated glass coverslips (12-mm diameter; Bellco, Vineland, NJ). Terminal differentiation of hES-NP cells was induced in ITS+AA medium by withdrawing bFGF in the presence or absence of BDNF (20 ng/ml; R&D Systems), GDNF (20 ng/ml; R&D Systems), and dibutyryl cAMP (0.5 mmol/l; Sigma).

Retroviral transduction. The retroviral vectors expressing LacZ (control), Bcl-XL, and SHH were constructed as described previously.40 The bicistronic vector pSHH-IRES-Bcl-XL, designed for the simultaneous expression of SHH and Bcl-XL in the transduced cells, was constructed by replacing LacZ of pSHH-IRES-LacZ with the Bcl-XL complementary DNA fragment amplified by PCR. The retroviral vectors were introduced into the retrovirus packaging cell line 293gpg by transient transfection with Lipofectamine (Invitrogen). After 72 hours, the supernatants were harvested. The cells were exposed to viral supernatant for 2 hours in the presence of polybrene (1 µg/ml; Sigma).

Immunofluorescent staining. Cultured cells and cryosectioned brain slices were fixed in freshly prepared 4% paraformaldehyde in PBS, and blocked with 5% normal goat serum and 0.1% Triton X-100 in PBS at room temperature for 1 hour. In case of detecting human TH+ neurons in transplanted striatum, different antigen retrieval procedures, such as SDS treatment or boiling, were employed before the blocking procedure as follows. Sectioned brain slices were treated with 1% SDS in PBS at room temperature for 5 minutes and then immersed in PBS.41,42,43,44 For antigen retrieval by boiling, slices were immersed in 10 mmol/l citrate buffer, pH 6.0, and boiled for 15 minutes in a microwave oven, then chilled on ice for 30 minutes (refs. 45,46,47). The following primary antibodies were used: nestin no. 130 (1:100; Martha Marvin and Ron McKay, National Institutes of Health, Bethesda, MD); TH (1:250; Pel-Freez, Rogers, AR; 1:200; ImmunoStar, Hudson, WI; 1:1,000; Sigma); cleaved caspase-3 (1:100; Cell Signaling Technology, Beverly, MA); green fluorescent protein (1:2,000; Invitrogen); Nurr1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA); Girk2 (1:100; Alomone Labs, Jerusalem, Israel); Pax2 (1:100; Covance, Richmond, CA); vesicular monoamine transporter 2 (1:500; Pel-Freez); HN (1:100; Chemicon, Temecula, CA); Ki67 (1:100; Novocastra, Newcastle, UK); proliferating cell nuclear antigen (1:40; Upstate Biotechnology, Lake Placid, NY); phospho-Histone H3 (1:100; Upstate, Temecula, CA); Engrailed-1 (1:50; Developmental Studies Hybridoma Bank, Iowa City, IA); Pax5 (1:50; BD Biosciences, Franklin Lakes, NJ); neuron-specific class III β-tubulin (TuJ1; 1:500; Covance); microtubule-associated protein 2 (MAP2, 1:200; Sigma); HuC/D (1:100; Chemicon); Oct3/4 (1:100; Santa Cruz Biotechnology). Appropriate fluorescence-tagged secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for visualization. Cells and tissue sections were mounted in VECTASHIELD containing 4′,6 diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and analyzed under an epifluorescence (Nikon, Tokyo, Japan) or a confocal (Leica PCS SP5; Leica Microsystems Heidelberg, Heidelberg, Germany) microscope.

Propidium iodide staining and lactate dehydrogenase assay. Cultured cells on glass coverslips were stained with propidium iodide (50 µg/ml; Roche Diagnostics, Germany), fixed in freshly prepared 4% paraformaldehyde in PBS, and mounted in VECTASHIELD containing 4′,6 diamidino-2-phenylindole. Lactate dehydrogenase activity was measured with a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI) according to the manufacturer's instructions. Results were expressed as a percentage of maximum lactate dehydrogenase release obtained on complete cell lysis following 1.0% Triton X-100 treatment. Fresh ITS medium was used as the negative control (0%).

In vivo transplantation and histological procedure. Animals were housed and treated in accordance with guidelines from the National Institutes of Health. Female Sprague–Dawley rats (200–250 g) were lesioned by unilateral stereotactic injection of 6-hydroxydopamine (6-OHDA; Sigma) into the substantia nigra and the median forebrain bundle as described previously.6 Four weeks later, the animals were tested for drug-induced rotational asymmetry (amphetamine 3 mg/kg i.p.; Sigma). Rotation scores were monitored for 60 minutes in an automated rotameter system (Med Associates, St Albans, Vermont). Animals with ≥5 turns/minute ipsilateral to the lesion were selected for transplantation. For transplantation, hES-NP cells expanded with bFGF were harvested and dissociated into single cells. Three microliters of cell suspension (2.5 × 105 cells/µl in PBS) per site were deposited at two sites of the striatum [coordinates in anteroposterior, mediolateral, and ventral relative to bregma and dura: (i) 0.07, −0.30, and 0.55; (ii) −0.10, −0.40, and −0.50; incisor bar set at 3.5 mm using a 22-gauge needle]. The needle was left in place for 5 minutes following each injection. Control sham-operated rats were injected with a vehicle (PBS). The rats received daily injections of cyclosporin A (10 mg/kg, i.p.) starting 24 hours prior to grafting and continuing for 3 weeks, followed by a reduced dose (5 mg/kg) for the remaining time. At 2, 4, 6, and 8 weeks after transplantation, amphetamine-induced rotations were determined. Eight weeks after transplantation, the animals were anesthetized with phenobarbital and perfused transcardially with 4% paraformaldehyde in PBS. Brains were equilibrated with 30% sucrose in PBS and sliced on a freezing microtome (CM 1850; Leica, Wetzlar, Germany). Free-floating brain sections (35 µm thick) were subjected to immunohistochemistry as described above. TH-immunoreactive cells in the graft were counted by analyzing every sixth uniform section (35 µm thick) with Abercrombie correction.48 The total number of TH-immunoreactive cells in the graft was estimated by multiplying the sum of TH+ cell counts by six. Graft areas were determined using an image analyzer (Analysis version 3.2; Soft Imaging System, Munster, Germany), and the Cavalieri estimator was used to calculate graft volumes. Hematoxylin and eosin staining was performed.

Reverse transcription–PCR. Total RNA preparation, cDNA synthesis, and reverse transcription–PCR reactions were performed as described previously.6 Information on primer sequences and optimum conditions will be provided upon request.

Cell counting and statistical analysis. Uniform random sampling procedure was used for cell counts and quantified using the fractionation technique.49 Immunoreactive cells on coverslip cultures were counted on randomly selected microscopic fields in a region of uniform cell growth using an eyepiece grid at a final magnification of ×200 or ×400. On each coverslip, 10–20 microscopic fields were counted, and 2–4 coverslips were analyzed in each experiment. Data are expressed as mean ± SEM of at least three independent experiments. Statistical comparisons were made by analysis of variance, with Scheffé or Tukey post hoc analysis (SPSS 15.0; SPSS, Chicago, IL) when two or more groups were involved. The paired t-test was applied for comparison of TH+ cells detected using the immunostaining method with or without SDS treatment in identical sections of hES-derived grafts.

SUPPLEMENTARY MATERIALFigure S1. Midbrain DA neuron properties of TH+ cells differentiated from hES-NP cells. a, RT-PCR analysis of genes specific for general (TH, DAT, VAMT2, AADC) and midbrain (Nurr1, En1, Ptx3, Lmx1b, Girk2) DA neurons in cultures of undifferentiated hES (hES) and differentiated hES-NP cells at NP passage 2 (P2), P5, and P8. b-j, Immunocytochemical data showing expression of neuron-specific TuJ1 (b), MAP2 (c), HuC/D (d), mature DA neuron-specific VMAT2 (e), midbrain DA neuron-specific En1 (f), Nurr1 (g), and GirK2 (h) in TH+ cells differentiated from hES-NP cells. Co-labeled cells are indicated as arrows. Note that the midbrain specific markers, Pax2 and Pax5, are expressed, but not localized in TH+ cells in differentiated hES-NP cultures (i, j). Scale bar, 40 um. RT-PCR and immunocytochemical analyses were performed 15 days after differentiation of hES-NP cells at NP passages 5-6.Figure S2. Raw data for amphetamine-induced rotation scores for 1 h obtained from individual animals grafted with early NP (a), B+H early NP (b), late NP (c) and B+H late NP (d).

Supplementary Material

Figure S1.

Midbrain DA neuron properties of TH+ cells differentiated from hES-NP cells. a, RT-PCR analysis of genes specific for general (TH, DAT, VAMT2, AADC) and midbrain (Nurr1, En1, Ptx3, Lmx1b, Girk2) DA neurons in cultures of undifferentiated hES (hES) and differentiated hES-NP cells at NP passage 2 (P2), P5, and P8. b-j, Immunocytochemical data showing expression of neuron-specific TuJ1 (b), MAP2 (c), HuC/D (d), mature DA neuron-specific VMAT2 (e), midbrain DA neuron-specific En1 (f), Nurr1 (g), and GirK2 (h) in TH+ cells differentiated from hES-NP cells. Co-labeled cells are indicated as arrows. Note that the midbrain specific markers, Pax2 and Pax5, are expressed, but not localized in TH+ cells in differentiated hES-NP cultures (i, j). Scale bar, 40 um. RT-PCR and immunocytochemical analyses were performed 15 days after differentiation of hES-NP cells at NP passages 5-6.

Click here for supplemental data (2.5MB, tiff)
Figure S2.

Raw data for amphetamine-induced rotation scores for 1 h obtained from individual animals grafted with early NP (a), B+H early NP (b), late NP (c) and B+H late NP (d).

Click here for supplemental data (76.4KB, tiff)

Acknowledgments

This work was supported by Stem Cell Research Center of the 21st Century Frontier Research (SC4150) and Medical Research Center (R13-2008-026-01000-0) programs funded by the Ministry of Science and Technology, Republic of Korea.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Midbrain DA neuron properties of TH+ cells differentiated from hES-NP cells. a, RT-PCR analysis of genes specific for general (TH, DAT, VAMT2, AADC) and midbrain (Nurr1, En1, Ptx3, Lmx1b, Girk2) DA neurons in cultures of undifferentiated hES (hES) and differentiated hES-NP cells at NP passage 2 (P2), P5, and P8. b-j, Immunocytochemical data showing expression of neuron-specific TuJ1 (b), MAP2 (c), HuC/D (d), mature DA neuron-specific VMAT2 (e), midbrain DA neuron-specific En1 (f), Nurr1 (g), and GirK2 (h) in TH+ cells differentiated from hES-NP cells. Co-labeled cells are indicated as arrows. Note that the midbrain specific markers, Pax2 and Pax5, are expressed, but not localized in TH+ cells in differentiated hES-NP cultures (i, j). Scale bar, 40 um. RT-PCR and immunocytochemical analyses were performed 15 days after differentiation of hES-NP cells at NP passages 5-6.

Click here for supplemental data (2.5MB, tiff)
Figure S2.

Raw data for amphetamine-induced rotation scores for 1 h obtained from individual animals grafted with early NP (a), B+H early NP (b), late NP (c) and B+H late NP (d).

Click here for supplemental data (76.4KB, tiff)

Articles from Molecular Therapy: the Journal of the American Society of Gene Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

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