Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum-F

干细胞之家 - 中国干细胞行业门户第一站 › 干细胞之家论坛 › 干细胞文献资源库 › 《stemcells》2004-2005文献 › Differentiation of Human Embryonic Stem Cells to Dop ...



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 24 下一页

返回列表

查看: 267619\|回复: 233	go Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum-F

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

发表于 2009-3-5 10:35 |显示全部帖子

a BresaGen Inc., Athens, Georgia, USA;
; X6 J+ w  Q6 d3 ?. C7 _6 a
b Department of Genetics, University of Georgia, Athens, Georgia, USA;
2 ^# E/ ?( |- b- {
c Institute of Molecular Medicine and Genetics and
& G, o% ^$ U# a! P
d Departments of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia, USA;
& [, Y; `# P8 [
e BresaGen Ltd., Thebarton, Adelaide, Australia;% U5 F9 D* S. f7 O0 ?8 n4 u

f Medical Research Service, Augusta Veterans Affairs Medical Center, Augusta, Georgia, USA) l* v/ E3 M& v" D$ h

Key Words. Embryoid bodies ? ES cells ? Differentiation ? Neural differentiation ? Serum-free medium ? Real-time RT-PCR

Correspondence: Thomas C. Schulz, Ph.D., BresaGen Inc., 111 Riverbend Rd., Athens, Georgia, 30605, USA. Telephone: 706-613-9878; Fax: 706-613-9879; e-mail: tschulz@novocell.com; and Brian G. Condie, Ph.D., Department of Genetics, Life Sciences Building, University of Georgia, Athens, GA 30602, USA. Telephone: 706-542-1431; Fax: 706-583-0691; e-mail: bcondie@uga.edu
4 j7 n" ?+ w; B: Q/ {
ABSTRACT/ }$ p' i, M' p0 s

The use of human embryonic stem cells (hESCs) as the source of neural cells for transplantation therapies has several advantages . hESCs are a source of many well-characterized human stem and progenitor cell types that correspond to the cells found in a developing embryo. Recent studies have documented important advances in the culture of hESCs for cell therapy. This work indicates that it will be possible to propagate normal and undifferentiated hESCs without feeder cells in highly defined and controlled culture conditions, allowing the generation of master cell banks to support cell transplants . In addition to the ability to propagate and expand hESCs in well-defined conditions, it will be important to develop methods to differentiate the cells using simple and well-defined culture conditions. Ideally, differentiation should be carried out in a serum-free environment using approaches that can be easily scaled for the production of large numbers of differentiated cell types. The cells cultured in these conditions should respond to known developmental modulators in a way predicted from their normal function in vivo or shown in other differentiation studies of hESCs or nonhuman embryonic stem (ES) cells.
. ~# [; A6 i6 X" A# }- W
Previous studies have shown that grafts of fetal midbrain dopaminergic neurons could survive, reinnervate, and function in patients with Parkinson’s disease (PD) and provide a proof of principal for this approach . However, two double-blind controlled trials revealed significant issues that need to be addressed before larger trials are warranted . One concern is the difficulty in standardizing the fetal midbrain tissue used for implantation, which will be critical for consistent clinical outcomes. The generation of midbrain dopamine neurons from hESCs would provide cell populations that could be expanded, characterized, and standardized in vitro, providing optimal populations for studies in animal models of PD . This approach would also provide a useful model for many aspects of human neurogenesis, including examination of the molecular and developmental controls of the midbrain lineage and functional analyses of their cellular and physiological characteristics. Several methods to generate midbrain dopaminergic neurons from mouse and primate ES cells currently exist, some of which have led to recovery of symptoms in rat models of PD after cell implantation . Many of these rely on complex multistep protocols requiring the exposure of the cells to stromal cell lines , multiple growth factors , or expression of transgenes such as Nurr1 . The utility of these methods to direct differentiation from hESCs has not yet been reported, and use of cocultures, added signaling factors, or transgenic approaches could lead to additional regulatory as well as cell production barriers that will complicate their use in eventual therapies and increase their cost.
8 B# `. w! x/ t. J! e- ]
We have previously reported effective neural differentiation of hESCs in serum-free conditions under the influence of the HepG2-conditioned medium MedII . In this study we report the differentiation of hESCs to midbrain dopaminergic neurons in a simple serum-free suspension system. This occurred in the absence of added growth factors or neural-inducing agents, demonstrating that it was driven by signaling within suspension aggregates. We showed that this differentiation was initially inhibited by bone morphogenic protein (BMP)-4, but later BMP signaling induced peripheral neuronal differentiation. These effects of BMP-4 were the same as those previously observed in cultures of mouse or nonhuman primate ES cells, demonstrating that cell fates can be easily manipulated by the addition of exogenous factors in our culture system. The differentiated tyrosine hydroxylase–positive (TH ) neurons were susceptible to 6-hydroxy-dopamine (6-OHDA), plated cultures released dopamine and other catecholamines upon depolarization, and surviving TH  neurons were detected 8 weeks after transplantation to the 6-OHDA–lesioned rat brain. Our approach represents a simple and potentially scalable platform for the large-scale derivation of dopaminergic neurons for studies in animal models of PD and the molecular, cellular, and physiological examination of this differentiation pathway.4 O- ~9 `- E. }! N

MATERIALS AND METHODS
; Y7 W9 d( t+ o/ o6 [
Collagenase/Trypsin Passaging and SSEA-4 Enrichment of hESCs+ J8 i, S+ N  j( R+ d& N! \) v
& g6 c% b9 `' x
The BG01 and BG03 hESC lines  were used in this study and are listed on the NIH registry. Until the time of this study, these cells had been maintained exclusively by manual microdissection of individual undifferentiated colonies (microdissection passaging). Because of the ability to selectively passage morphologically undifferentiated cells, microdissection passaging is currently the most appropriate method to maintain long-term cultures of undifferentiated hESCs and may contribute to the maintenance of a normal karyotype . However, this approach is laborious, and scaling up cultures for experiments is difficult. Therefore, we tested several enzymatic cell dissociation methods for maintaining and expanding BG01 cells. After cell dissociation with collagenase and trypsin, undifferentiated BG01 cells were enriched by immunomagnetic-bead cell sorting using a monoclonal antibody against SSEA-4, a cell-surface antigen that is robustly expressed on pluripotent hESCs . Flow cytometric analysis of a representative experiment detected SSEA-4 expression on 85% of the starting population of cells, whereas 99.2% of the cells expressed SSEA-4 after immunomagnetic enrichment, with 60.7% of cells in the flow-through being SSEA-4 . Cultures enriched for SSEA-4–expressing cells (Fig. 1A) grew as colonies that strikingly resembled those of mouse ES cells and other hESC lines passaged with trypsin  and exhibited the characteristic profile of the following pluripotent markers expressed by hESCs: SSEA-1–, SSEA-3 , SSEA-4 , Tra-1-60  (Figs. 1B–1E, respectively), Tra-1-81  (not shown), and OCT-4  (Fig. 1F). In addition, aggregates of SSEA-4  cells allowed to differentiate in a serum-containing medium formed cells expressing ectodermal (Nestin, Sox1), endodermal (Amylase, AFP), and mesodermal (Cardiac actin) markers, suggesting that the SSEA-4–enriched cells could form lineages of the three embryonic germ layers (not shown) and maintained their pluripotency. These hESCs also expressed the neural progenitor markers nestin (Fig. 1F) and vimentin (Fig. 1G). Expression of vimentin has been detected in the H1 hESC line by RT-PCR and immunocytochemistry , whereas RT-PCR has detected nestin expression in some lines but not others . It is possible that nestin is expressed in at least a subset of the cells within most other established hESC lines./ `8 [5 e/ q8 ?9 t9 k. f, v

Figure 1. Culture and neural differentiation of hESCs. (A): Collagenase/trypsin-passaged and SSEA-4–enriched BG01 hESCs. BG01 cells were SSEA-1– (B), SSEA-3  (C), SSEA-4  (D), Tra-1-60  (E), OCT-4  and Nestin  (F), and Vimentin  (G). (H): ?III tubulin and (I) TH immunostaining of plated MedII/FGF2 differentiations. (J): Merged image of (H, I). TH (K), vesicular monoamine transporter 2 (L), and merged (M) immunostainings of plated MedII–/FGF2  differentiations show cell body staining. Scale bars = 100 μm (A, H–J) and 50 μm (B–G, K–M). Abbreviations: DAPI, 4',6'-diamidino-2-phenylindole; FGF2, fibroblast growth factor 2; hESC, human embryonic stem cell; TH, tyrosine hydroxylase .

To produce the numbers of cells required for these studies in a timely fashion, we used cultures of BG01 cells maintained by collagenase/trypsin dissociation and SSEA-4 enrichment unless otherwise noted. Because of the possibility of accumulating aneuploidies as well as spontaneously differentiated cells in enzymatically passaged BG01 cultures, these cells were not used beyond approximately 20 passages after SSEA-4 enrichment or 30 total passages with collagenase/trypsin. Chromosome counting indicated that under these culture conditions, up to 50% of cells had an abnormal karyotype after a total of 33 passages with collagenase/trypsin (I. Nasonkin, unpublished data). Key experiments (derivation and proliferation of neural progenitors in DMEM/N2, the generation of large networks of TH  cells but rare D?H  cells in suspension aggregates, and evoked release of dopamine) were confirmed using karyotypically normal BG01 and BG03 cells maintained by passaging as clumps of cells, either with microdissection passaging or disaggregation using EDTA-free trypsin.0 g2 R7 E( {- ~8 p$ m

Neural Differentiation of hESCs in a Serum-Free Minimal Medium
3 @+ z$ [6 \( V3 j6 d& A7 j
A summary of the neural and dopaminergic differentiation observed in these experiments is outlined in Table 2. We performed differentiation experiments using variations of two basic conditions: 50% MedII-conditioned medium plus FGF2 (DMEM/F12 N2 MedII FGF2) and DMEM plus N2 supplement (minimal medium). Experiments were typically analyzed after 1 month in suspension, and both of these conditions supported the differentiation of large networks of TH  neurons. Because initial survival of cell aggregates was lower in DMEM/N2 conditions, we also derived cell aggregates into MedII/FGF2 for 3–5 days, followed by 1 month in minimal medium, which also generated large networks of TH  neurons. We used minimal DMEM/N2 conditions as a base to assess the role of additional factors on neural differentiation. Finally, for some analyses, differentiated aggregates were plated to adherent culture for approximately 1–2 weeks in either MedII/FGF2 or Neurobasal medium supplemented with B27, serum, BDNF, and GDNF, because minimal DMEM/N2 conditions did not support effective attachment of differentiated aggregates to adherent culture.

Table 2. Summary of neural differentiation experiments% _- K" A% ~9 ?* |& S4 v7 h" L

We initially tested the ability of the collagenase/trypsin–passaged and SSEA-4–enriched BG01 cells to differentiate in serum-free conditions in MedII/FGF2 medium . MedII-conditioned medium has been shown previously to promote neural differentiation from mouse, rhesus monkey, and human embryonic stem cells . Whole hESC colonies were removed from the feeder layer and cultured in suspension. Characteristic folds and rosettes of neural precursors were observed after 5–10 days of culture, as observed in differentiations performed from microdissection-passaged hESCs . Cell aggregates were plated on polyornithine/laminin-coated chamber slides 2 or more weeks after derivation and cultured for an additional 5–7 days before immunostaining. Stained cultures were highly enriched for nestin  neural precursor rosettes and large networks of ?III tubulin  (Fig. 1H) neurons. Most of these neurons also expressed TH (Figs. 1I, 1J). Scoring of isolated ?III tubulin–expressing neurites in merged images showed that approximately 75% (69 of 90, n = 5 fields) were TH /?III tubulin . This was strikingly different from our previous differentiations from microdissection-passaged hESCs , in which suspension cultures were plated after only approximately 1 week of culture and previous reports , in which TH  neurons were rare. MedII seemed to enhance rather than induce neuronal differentiation, because significant differentiation to TH  and VMAT2  neurons also occurred in the same medium without added MedII (Figs. 1K–1M).  i% u# d" @& U- W# n+ S9 v
$ K( A  Q( @* @
The ability of BG01 cell aggregates to differentiate into neurons in serum-free suspension culture led us to test the role of the added MedII and FGF2 in promoting early neural lineage formation. In these experiments, hESC aggregates were cultured in DMEM/N2. Unlike FGF2/MedII differentiations, aggregates incubated in DMEM/N2 exhibited a very high level of obvious cell death through their first approximately 2 weeks, indicating that MedII/FGF2 contributed significantly to cell survival. This was consistent with our previous results, indicating that MedII provided a cell survival/proliferation activity rather than a neural inducing factor . Only hESC aggregates that were initially larger than approximately 150 μm were viable and proliferated in the minimal medium, suggesting a community effect in the delivery of essential growth factors and signaling within differentiating aggregates. After differentiation for 2 weeks in DMEM/N2, aggregates seemed to be comprised largely of neural precursor rosettes/neurectoderm structures (Fig. 2A). As suspension aggregates were cultured further, there appeared to be a gradual loss of this distinct morphology, from approximately 2–4 weeks, possibly indicating a shift away from neural progenitor proliferation to neuronal differentiation (not shown). However, persistence of neural precursor rosettes could be detected even after 4 weeks of differentiation. Sectioning, followed by toluidine blue or DAPI staining (Fig. 2B), demonstrated that at 2 weeks, cell aggregates cultured in DMEM/N2 were comprised of distinctly organized regions of neural precursor rosettes and nonrosette regions. Counts of DAPI-stained nuclei (Fig. 2B, inset) indicated that rosette neural progenitor structures comprised 39.4 ± 12.3% (14334/36663 nuclei, n = 11 sections) of the cells. The nonrosette regions were demonstrated by whole-mount analysis and counting of anti-HuC/D and DAPI-stained overlayed images to contain 45.5 ± 7.2% HuC/D  early postmitotic neurons (445/984 cells, n = 5 fields). DMEM/N2 aggregates were also dense with ?III tubulin  neuronal extensions (Fig. 2C) and TH  neurons (Fig. 3A). The rosettes exhibited a characteristic structure with a core of tightly packed proliferating neural precursor cells, of which 7.3 ± 4.4% (30 of 376, n = 7 fields) exhibited condensed mitotic chromosomes when DAPI-stained nuclei in 1-μm confocal optical sections were counted. Rosette cells expressed nestin (Figs. 2E, 2G) and vimentin (Figs. 2F, 2G), whereas expression of HuC/D, a marker of early postmitotic neurons , was first observed in the differentiating cells surrounding the rosettes (Figs. 2H–2J). Double immunostaining of plated cultures with HuC/D and phospho-HistoneH3, a mitotic marker , was used to confirm the postmitotic status of the neurons associated with neural precursor rosettes, with no phospho-H3 /HuC/D  cells being observed from >500 counted HuC/D  cells (Figs. 2K–2M). Expression of synapsin (Fig. 2N) and synaptophysin (Fig. 2O) was detected in plated neurites, suggesting the formation of synaptic complexes. In addition to the analysis of the cell aggregates with immunocytochemistry, the expression of general neuron markers as well as markers of neurotransmitter phenotypes was determined by RT-PCR analysis and a focused microarray screen. We analyzed gene expression in BG01 DMEM/N2 suspension aggregates after 6 weeks of differentiation using a focused array of 266 human genes, selected to represent different human stem cell populations . We compared gene expression in hESCs and in differentiated aggregates (Fig. 4) and found 14 transcripts that were upregulated in the differentiated cells. Many of these genes have known or presumed function during neural development and differentiation, including BMP signaling (BMPR2), FGF signaling (FGF11, FGFR1, FGFR2), WNT signaling (FZD3), neurogenic functions (CXCR4, DLK1, VEGF), and neurotrophin signaling (NTRK2). Of the 11 SOX-family transcription factors present on the array, only SOX1, 2, 3, and 4, which exhibit neural tube/progenitor expression or function, were detected. Common markers of neuronal cell function were also upregulated such as neurofilaments (INA, NEFL), MAP2, and NCAM1. The expression of FGF11 confirmed that the differentiated aggregates contained neuronal progenitors. In a previous analysis of rat central nervous system (CNS) progenitors, it was found that FGF11 expression was activated after neuronal precursors appeared within the CNS, and cell sorting of the progenitors showed FGF11 expression exclusively within the E-NCAM  neuronal progenitor population . In addition, the focused array contained more than 22 markers of differentiated nonneural lineages representing endoderm, mesoderm, and nonneural ectoderm. Expression of most of these markers (20 of 22) was not detected (Fig. 4), confirming enriched neural differentiation in these aggregates. A previous characterization of the sensitivity of similar focused microarrays showed a 96% correspondence between the results of the arrays and RT-PCR analysis . This shows that these focused micro-arrays are quite sensitive because of the use of gene-specific primers in making the cDNA probe. The overall pattern of expression in BG01 hESCs using this array was similar to that reported previously . Transcripts that were upregulated in hESCs were CER1, FGF2, DNMT3B, FOXM1, FZD7, ITGA6, PDGFA, POU5F1, and TERF1. RT-PCR analysis of DMEM/N2 suspension aggregates at 4 weeks detected expression of choline acetyltransferase, vesicular glutamate transporters 1, 2, and 3, and the vesicular inhibitory amino acid transporter (not shown), which are markers of cholinergic, glutaminergic, and GABAergic/glycinergic neurons, respectively. The expression of GAD67 was not detected by immunostaining or RT-PCR analysis, suggesting that few -aminobutyric acid (GABA)–producing neurons were present. The capacity for glial differentiation was demonstrated by the expression of GFAP (Fig. 2P, inset). This analysis suggested that a range of neural lineages could be generated in this system.
6 i9 H0 W+ F7 k' J
Figure 2. Neural differentiation of DMEM/N2 suspension aggregates. (A): Suspension DMEM/N2 aggregates after 2 weeks. Inset shows a higher magnification indicating a central cavity (*), surrounded by the radial organization of the neuroepithelia. (B): Three-μm plastic section of DMEM/N2 aggregates stained with toluidine blue or DAPI (inset), showing neural precursor rosettes (arrows) and nonrosette regions (*). (C): Whole-mount ?III tubulin immunostaining and 1-μm confocal optical section of suspension DMEM/N2 aggregates. Rosette area is indicated (*). (D): Whole-mount DAPI staining and 2-photon, 1-μm optical confocal section of DMEM/N2 aggregates. Condensed chromosomes in the core regions of rosettes (arrows) and two adjacent rosettes (dashed lines) are indicated. (E): Nestin expression in neural rosette cells. The arc of a rosette is indicated (dashed lines). (F, G): Coexpression of Vimentin and nestin in neural rosette cells. (H–J):Whole-mount HuC/D immunostaining and DAPI staining of DMEM/N2 aggregates. (H): DAPI staining (in grayscale) showing a neural precursor rosette (dashed oval). (I, J): HuC/D was expressed in the cells immediately surrounding the rosette structures. (K–M): Immunostaining of plated DMEM/N2 aggregates demonstrated that rosette-associated (dashed oval) early neurons were postmitotic, with no double-positive (K) phospho-HistoneH3 and (L) HuC/D cells detected (M). Synapsin (N), synaptophysin (Synapt.) (O), and GFAP (P) expression in plated cultures is shown. (P): Inset, reverse transcription–polymerase chain reaction of GFAP expression in DMEM/N2 suspension aggregates. (Q, R): DMEM/N2 differentiation-derived neurons plated in MedII/FGF2 possess the physiological characteristics of central nervous system neurons. (Q): Leak-subtracted current (I) traces evoked by a family of increasingly depolarizing voltage (V) commands (–50, –30, –10,  10 mV) from a holding potential of –70 mV are shown superimposed. Inward and outward currents characteristic of sodium and delayed-rectifier potassium currents were evoked in 9 of 10 cells. (R): Inward membrane current and an increase in noise evoked by application of 1 mM glutamate (indicated by horizontal bar); holding potential was –70 mV. Similar currents were evoked in 10 of 10 cells. Scale bars = 100 μm (A, B, C, N), 50 μm (B inset, H–J, K–M), and 25 μm (D, E, F, G, P, O). Abbreviations: DAPI, 4',6'-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; GFAP, glial fibrillary acidic protein.3 Z7 E) U' x6 k# p, C1 \
: F6 E2 `7 ~2 ]+ ?, ]+ @! d
Figure 3. BMP-4 and serum affect neural and dopaminergic differentiation. BG01 hESC aggregates were differentiated under different conditions and examined after 1 month. (A–C): BMP-4 inhibits neuronal differentiation of hESCs. (A): DMEM/N2 aggregates and parallel cultures containing (B) 10 ng/ml BMP-4 or (C) 10 ng/ml BMP-4 and 10% fetal calf serum generated 180, 18, and ~300 viable aggregates 11 days after derivation, respectively. Aggregates were immunostained with ?III tubulin and TH, demonstrating dense neuronal networks (A) and nearly complete inhibition of neuronal differentiation (B).An example of maximal neuronal differentiation in  BMP-4 conditions is shown, with other aggregates exhibiting no neurons. (C): Recovery and enhanced overall aggregate viability, but neuronal differentiation was not restored. (D–F): Addition of BMP-4 to DMEM/N2 differentiations from days 5 through 9 induced peripheral neuronal differentiation. (D): Rare peripherin  cells in DMEM/N2 differentiations. (E): High proportions of ?III tubulin /TH  cells generated with d5-9 BMP-4 treatment, but (F) large proportions of peripherin  cells were induced. (G–I): Serum inhibits dopaminergic differentiation. Differentiations in 10% serum showed an increase in nonneural differentiation (G, H) but still generated a large number of ?III tubulin  neurons. Only rare TH  neurons were observed. (I): RT–polymerase chain reaction for LMX1B expression in DMEM/N2 and 10% serum conditions. Scale bars = 100 μm (A–C, G) and 50 μm (D–F, H). Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; hESC, human embryonic stem cell; RT, reverse transcription; TH, tyrosine hydroxylase.& V: z1 K0 ^" e2 q' p9 D

Figure 4. Focused array of gene expression in BG01 hESC and DMEM/N2 suspension aggregates differentiated for 6 weeks. The spots and names of the transcripts that were upregulated in each condition are indicated (arrows). The bottom row shows the indicated control cDNAs. Marker expression in DMEM/N2 differentiations: SOX genes expressed: SOX1,2,3 (neural tube/progenitors), SOX4 (differentiating neural progenitors, heart, B cells). SOX genes not detected: SOX5,6,10,13,15,17,18 (chondroblasts, neural crest, kidney, ovary, embryonic artery, testis, definitive endoderm, heart). Markers of nonneural lineages that were not detected: AFP, MYH11, CDH3,5,15, FABP4,6, GATA4, GCG, INSRR, ISL1, KRT8,14,15,17, MYH6, MYL4, NKX2.5, PECAM1, TNC (yolk sack, liver, smooth muscle, placenta, mammary gland, vascular, endothelia, muscle, adipose, enterocyte, heart, gut, epithelia, pancreas, kidney, islet, platelets, endothelial cells, mesenchyme, cartilage, bone). Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; hESC, human embryonic stem cell.; l: ?* E% @- N! |  a
8 D6 A( `; W; r. R  P8 D" s
To physiologically verify the phenotype of hESC-derived neurons, whole-cell voltage-clamp recordings were made from DMEM/N2 differentiations plated to adherent culture in MedII/FGF2 medium. Depolarizing voltage commands from a negative holding potential evoked rapid inward sodium currents and delayed outward potassium currents (n = 9 of 10 cells; Fig. 2Q). Application of the excitatory and inhibitory neurotransmitters glutamate (Fig. 2R) and GABA (not shown) evoked rapidly desensitizing membrane currents consistent with the expression of ionotropic glutamate and GABA receptors (n = 10). Therefore, these neurons expressed the voltage- and ligand-gated ion channels that would allow them to generate action potentials and receive synaptic information.* |3 k9 r: ]+ S$ W5 E" |
  {0 `: s. L7 F! }# \% c; |+ a
Early Exposure to BMP-4 Antagonizes Neuronal Differentiation and Later Exposure Induces Peripheral Neurons' K8 ]3 s+ ]8 r& t* W4 L& t
! }8 h) J9 L/ o" T: k  y
To demonstrate that the cell aggregates cultured in minimal medium would respond to extracellular factors, we tested the effect on neural differentiation of early or late exposure to BMP-4. We tested the ability of BMP signaling to antagonize the formation of neuronal lineages in hESC aggregates cultured in minimal medium. BMPs are a potent inhibitor of neural development and are known to induce nonneural ectoderm at the expense of neural ectoderm . We performed differentiations from parallel dishes of BG01 hESCs using three conditions: DMEM/N2 medium alone, DMEM/N2 BMP-4, and DMEM/N2 BMP-4 FCS. Addition of 10 ng/ml BMP-4 to the DMEM/N2 minimal medium led to an approximately 10-fold reduction in aggregate viability and nearly completely blocked the formation of ?III tubulin  and ?III tubulin /TH  neurons compared with aggregates in DMEM/N2 (Figs. 3A, 3B). Addition of serum to BMP-4–containing differentiations improved aggregate viability but did not restore the neural differentiation observed in DMEM/N2 conditions (Fig. 3C). These observations suggest that BMP-4 blocked neural lineage formation from the hESCs and instead stimulated the formation of nonneural serum-dependent cell types when cells were exposed to BMP-4 from day 1 of the differentiation. This was consistent with the known role of BMP-4 as an antagonist of neural lineage formation in Xenopus embryos and mouse ES cells . In later stages of neural development, BMP signals induce the formation of neural crest cells from the dorsal crest of the neuroepithelium . To examine the response of hESC differentiations to a later BMP signal, we added 10 ng/ml BMP-4 to DMEM/N2 differentiations from days 5 through 9, followed by culture in DMEM/N2 until 1 month after derivation. Unlike an early BMP signal, late exposure to BMP-4 did not affect the viability of aggregates. Whole-mount immunostaining using antibodies to TH, ?III tubulin, and peripherin, a marker of neural crest–derived peripheral neurons , detected a high proportion of ?III tubulin /TH  cells (Fig. 3E) but also a large number of peripherin  cells (Fig. 3F), indicating the presence of neural crest–derived neurons. In contrast, only rare peripherin  neurons were found in aggregates differentiated in DMEM/N2 (Fig. 3D), demonstrating that most of the ?III tubulin /TH  neurons represented a neural tube/CNS lineage.

We also examined the effect that addition of serum would have on differentiation within this system. In aggregates differentiated in DMEM/N2 plus 10% serum, large networks of ?III tubulin  neurons could still be detected after 1 month despite an increased amount of nonneural differentiation, such as cysts, compared with aggregates in DMEM/N2. The proportion of TH  neurons was greatly reduced in DMEM/N2 plus serum compared with DMEM/N2 (Figs. 3G, 3H). This indicated that although effective neuronal differentiation was possible in serum, factors present in these conditions may inhibit presumptive dopaminergic differentiation. Consistent with this, the midbrain dopaminergic marker LMX1B  was expressed at elevated levels in DMEM/N2 compared with serum-containing conditions (Fig. 3I).( }' H7 C# p7 o+ x

Neurons in the Cell Aggregates Express Multiple Markers Characteristic of Dopamine Neural Precursors and Neurons

Because large networks of TH  neurons were generated in DMEM/N2 conditions, but not in the presence of added serum, we examined gene expression in these conditions using multiple neural and dopaminergic markers. Real-time PCR was performed and gene expression was compared in differentiated aggregates after 4 weeks using GAPDH–normalized relative gene expression ratios (Table 3). Expression of SOX1  and MAP2 confirmed the presence of neural precursors and differentiated neurons, respectively, in both conditions. Higher expression of SOX1 in serum-free conditions and MAP2 in serum-containing conditions suggested that there was a bias toward proliferation of neural precursors in serum-free conditions and differentiation to neurons under the influence of serum. Several transcription factors that are involved in the specification of the midbrain dopaminergic lineage, EN1, NURR1, PITX3, and LMX1B, were all expressed at higher levels in serum-free conditions, at approximately 5.8-, 1.8-, 2.2-, and 1.5-fold, respectively . The difference in expression of NURR1 and PITX3 was statistically significant (p = .025 and .001, respectively), whereas the difference in EN1 (p = .081) expression was not significant in this analysis. The comparison of LMX1B expression was performed by densitometry of end-point RT-PCR (Fig. 3I). Analysis of markers of differentiated dopaminergic neurons demonstrated expression of TH, AADC, VMAT2, and the DAT in both conditions. Only AADC (p = .001) showed significant elevated expression in serum-free conditions, with VMAT2 not being significantly different and TH and DAT showing elevated expression in serum-containing conditions. The GIRK2 channel protein is a marker of A9 dopaminergic neurons , which are the major dopamine neuron subtype depleted in Parkinson’s disease . GIRK2 was expressed approximately 7.1-fold higher in serum-free conditions (p = .001). Expression of D?H, a more specific marker for other catecholaminergic neurons, was upregulated in 10% serum (p = .001). This expression analysis suggested formation of lineages expressing these markers in both conditions, with elevated expression of dopaminergic transcription factors and some markers of differentiated neurons in DMEM/N2 conditions. However, because this was a population-wide analysis, we also performed immunostaining to determine the relative distribution of neurons expressing some of these markers.
( i  Y# \7 y( j
Table 3. Real-time reverse transcription–polymerase chain reaction comparison of gene expression in aggregates differentiated in serum or serum-free conditions
& f6 Y( v5 N5 I1 }: X
To quantify the proportion of neurons in DMEM/N2 differentiations that expressed TH, aggregates were plated in adherent culture in MedII/FGF2 medium. Extensive networks of TH  neurons were observed (Figs. 5A, 5B) at a far greater abundance than reported previously . Scoring of isolated ?III tubulin  neurites in overlayed images showed that 73.9 ± 10.5% (46 of 64, n = 3 fields) were TH /?III tubulin  (Figs. 5A, 5B; panels 1, 2). To support the formation of mature neuron cell types, DMEM/N2 aggregates were plated in medium containing GDNF, BDNF, and 5% serum, a formulation known to support the survival of mouse ES cell–derived dopaminergic neurons . Counting of cell bodies demonstrated that TH  neurons comprised 63.8 ± 4.6% (689 of 1,085 cells, n = 3 wells) of the MAP2  population, whereas VMAT2  neurons comprised 94.9 ± 2.9% (317 of 334 cells, n = 3 wells) of the MAP2  population. Figure 5C shows an example of the most highly differentiated TH  neurons observed in these cultures, exhibiting a cell body, an approximately 580-μm dendritic extension and spines, and presumed growth cone. Additional immunostaining analysis demonstrated expression of additional markers of the dopaminergic phenotype in DMEM/N2 differentiations. Coexpression of the ?III tubulin, TH, VMAT2, and DAT proteins was demonstrated in aggregates in DMEM/N2 suspension cultures (Fig. 5D), which is similar to what was seen in MedII/FGF2 suspension aggregates (Fig. 5E). Coexpression of TH and active phospho-TH(Ser40) (Fig. 5F) , expression of the panneuronal marker TAU  and AADC (Fig. 5G), and coexpression of TH and DAT (Figs. 5H, 5I) were also demonstrated. Although RT-PCR analysis had detected D?H message in DMEM/N2 aggregates, expression was significantly lower than in 10% serum conditions. We used immunostaining to detect D?H-expressing cells in 4-week suspension aggregates. Only rare D?H  cells were detected in DMEM/N2 aggregates from trypsin-passaged BG01 cells, as well as from microdissection-passaged BG01 (Fig. 5J) and BG03 (Fig. 5K) cells that were differentiated with an initial 5 days in MedII/FGF2 followed by 1 month in DMEM/N2. These differentiations, as well as microdissection-passaged BG01 and BG03 that were differentiated in only DMEM/N2, also generated large networks of ?III tubulin /TH  neurons (Fig. 6A). We made several additional observations during the course of these experiments. Unlike embryoid body differentiations in serum, very few cysts were formed in embryoid bodies in serum-free conditions. Occasionally, pigmented epithelial cells were generated (Fig. 6B), similar to that observed in stromal cell–mediated differentiations of primate ES cells , although this was not a common event. RT-PCR and protein expression analyses therefore demonstrated the presence of the developmental and cellular factors that specify the midbrain dopaminergic lineage in suspension aggregates and mediate dopamine biosynthesis, vesicle loading, and dopamine reuptake after neurotransmitter release.: H4 ~0 S/ M2 U; n; }$ X3 D
" F+ g2 ?# c0 l9 b1 b5 E: d
Figure 5. Expression of dopaminergic markers in differentiated aggregates. Representative low-magnification images of DMEM/N2 aggregates plated in MedII/FGF2 medium and immunostained with (A) ?III tubulin and (B) TH. Boxed regions are shown at higher magnification in 1 and 2, with ?III tubulin, TH, and merged panels. (C):A highly differentiated MAP2 /TH  neuron observed when DMEM/N2 aggregates were plated in medium containing GDNF, BDNF, and 5% serum. Insets are higher magnifications of the indicated regions showing (left to right) presumed growth cone, connections to other MAP2  neurons, and cell body. The length of the extension from cell body to growth cone was ~580 μm. (D): Whole-mount four-color immunostaining of DMEM/N2 and (E) MedII/FGF2 suspension aggregates. ?III tubulin (visualized with a secondary antibody labeled with Alexafluor 350), TH (Alexafluor 594), VMAT2 (Alexafluor 647), and DAT (Alexafluor 488) images were merged to the cyan, magenta, yellow, and black channels of a CMYK image, respectively. Coexpression in cell bodies is indicated by white staining (arrows). (F–I): Immunostaining for markers of dopaminergic neurons in DMEM/N2 aggregates plated in MedII/FGF2 medium. (F): TH and phospho-TH(Ser40). (G): Aromatic amino acid decarboxylase and TAU. (H, I): DAT and TH. (J, K): Only rare D?H  cells were detected in suspension aggregates. D?H (arrow) and TH (J) and D?H (arrow) and ?III tubulin (K) immunostaining of microdissection-passaged BG01 and BG03 suspension aggregates, respectively. Scale bars = 100 μm (A, B), 50 μm (1, 2, C, F, G, J, K), 25 μm (E, H, I), 10 μm (D), and 5 μm (C insets). Abbreviations: BDNF, brain-derived neurotrophic factor; DAPI, 4',6'-diamidino-2-phenylindole; DAT, dopamine transporter; DMEM, Dulbecco’s modified Eagle’s medium; FGF2, fibroblast growth factor 2; GDNF, glial-derived neurotrophic factor; MAP2, microtuble-associated protein 2; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter 2.
) E7 ^5 K8 Z8 ]. ], y( D4 n
Figure 6. (A): Whole-mount TH and ?III tubulin immunostaining of BG03 suspension aggregates differentiated in DMEM/N2. (B): Pigmented cells observed in some BG01 DMEM/N2 suspension aggregates. (C): Differentiation of BG01 cells maintained on human keloid fibroblasts. Scale bars = 50 μm (B) and 25 μm (A, C). Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; TH, tyrosine hydroxylase.+ g$ r+ u0 U& w% J8 J/ @

The use of MEF feeder layers to support hESC culture will add regulatory complexity, because new clinical products derived using these feeder layers will be considered xenotransplants. Although others have demonstrated the maintenance of hESCs on human feeder cells  or in a feeder-free environment , it has not been determined whether hESCs grown under these conditions can differentiate to TH  neurons. We therefore differentiated collagenase/trypsin BG01 cells that had been maintained on a layer of human keloid fibroblasts (I.L., unpublished data) as DMEM/N2 aggregates and demonstrated that a high proportion of TH  neurons were also generated under these conditions (Fig. 6C). Therefore, hESCs that retain appropriate developmental potential may be able to be derived and maintained on human feeder layers, avoiding stringent xenotransplantation regulations.: S7 F% i0 N# s+ j

6-OHDA is a catecholamine neurotoxin that is taken up by dopaminergic cells expressing DAT and noradrenergic neurons . To examine whether the TH  neurons present in DMEM/N2 suspension aggregates were sensitive to 6-OHDA, we exposed aggregates to 10 mM or 1 mM 6-OHDA for 10 minutes, followed by a 5-hour recovery in MedII/FGF2 medium, so that degenerating cells could be visualized. Exposure to 6-OHDA led to widespread ablation of TH  neurons, which were rarely intact but exhibited disrupted and punctuated staining (Figs. 7A, 7B, 7D, 7E). ?III tubulin /TH– neuronal extensions and nonneuronal DAPI-stained nuclei appeared intact. Ascorbic acid–treated controls (not shown) were comparable with untreated aggregates (e.g., Fig. 3A). We used a 100- or 10-fold (Figs. 7C, 7F) excess of dopamine to compete with 6-OHDA uptake. This protected TH  neurons from ablation, indicating that the cells express functional dopamine or norepinephrine transporters.

Figure 7. Sensitivity to 6-OHDA and evoked release of catecholamines. (A–F): 6-OHDA selectively ablates TH  neurons in DMEM/N2 suspension aggregates. ?III tubulin and TH whole-mount immunostaining of DMEM/N2 aggregates exposed to 10 mM (A, D) or 1 mM (B, E) 6-OHDA. (C, F): Addition of 100-mM dopamine to 10-mM and 1-mM (not shown) 6-OHDA exposures protected TH  neurons from ablation. (G): HPLC traces demonstrating evoked release of 2175 pg/ml DA, 4475 pg/ml adrenaline (A), and 3404 pg/ml NA per 106 cells from DMEM/N2 differentiations plated in medium containing 5% serum, GDNF, and BDNF in response to depolarization with 56-mM KCl. These catecholamines were not detected in parallel cultures treated with HBSS. The elution times for adrenaline, NA, and DA were 7.13, 7.84, and 17.77 minutes, respectively. DHBA was used as an internal standard. The amplitude of electrochemical detection (mV) is shown for the HBSS and KCl samples. Scale bars = 100 μm (A–C) and 50 μm (D–F). Abbreviations: 6-OHDA, 6-hydroxydopamine; BDNF, brain-derived neurotrophic factor; DA, dopamine; DHBA, 3,4-dihydroxybenzyamine; DMEM, Dulbecco’s modified Eagle’s medium; GDNF, glial-derived neurotrophic factor; HBSS, Hanks’balanced salt solution; NA, noradrenaline; TH, tyrosine hydroxylase.

The release of dopamine in response to depolarization is a key indicator of the functional capacity of ES cell–derived neurons to synthesize dopamine, load it into vesicles, and release it in response to neurophysiological cues . BG01 hESCs were expanded by passaging as clumps with EDTA-free trypsin, followed by differentiation in serum-free suspension culture for 3 days in MedII/FGF2 and then DMEM/N2 until 1 month after derivation. Differentiated aggregates were then plated to adherent culture in medium containing GDNF, BDNF, and 5% serum, generating ~ 6.4 x 106 cells per 35-mm dish after 2 weeks. From these cultures, HPLC analysis detected evoked release of 2,175 pg/ml dopamine per 106 cells in response to a K -depolarizing stimulus (Fig. 7G). Release of 4,475 pg/ml adrenaline and 3,404 pg/ml noradrenaline per 106 cells could also be unambiguously resolved as peaks by HPLC. The evoked release of dopamine and other catecholamines was also detected in plated differentiations of microdissection-passaged hESCs, as well as collagenase/trypsin-passaged and SSEA-4–enriched BG01 hESCs, although in some experiments, only dopamine, but not adrenaline or noradrenaline, was detected (not shown). This indicates there is some variability in the proportions of these lineages that can be generated using these techniques. Although rare D?H  cells were detected in suspension aggregates, plating to adherent culture in medium containing BDNF, GDNF, and serum clearly supported the differentiation of adrenergic and noradrenergic lineages, as well as dopaminergic neurons.

Survival and Differentiation of Transplanted Human ES-Derived Neurons in the Striatum of a Rat Parkinson’s Disease Model% ]6 i6 i  C3 e1 x, S4 }

To determine whether TH  neurons derived from hESCs could survive engraftment, we transplanted differentiated aggregates into the striatum of rats with a unilateral lesion in the substantia nigra, which ablates the dopaminergic neurons projecting to the striatum. Aggregates that had been differentiated in MedII/FGF2 for 3 weeks, or DMEM/N2 for 1 month, were implanted into 8 and 15 rats, respectively, and rats were euthanized 8 weeks after implantation. Surviving cells were detected histologically (Fig. 8A) in 6 of 8 rats implanted with MedII/FGF2-differentiated aggregates and 11 of 15 implanted with DMEM/N2 aggregates. Biotinylated-human Alu repeat in situ hybridization probes were used as a lineage marker to confirm the presence of human cells (Fig. 8B). The implants varied considerably in size and the degree of cell survival, and one obvious teratoma containing cartilaginous structures and glandular epithelium was observed in a rat implanted with a DMEM/N2 cell population, indicating that some residual pluripotent cells may persist under these differentiation conditions. Survival of presumed neural rosettes was detected in some implants (Fig. 8A), and the expression of nestin (Figs. 8B, 8C) was also detected in many of the surviving implants. In some cases, regions of MAP2 expression were observed, and the expression of Ki67 indicated that proliferation was still occurring (data not shown). In implants of DMEM/N2-differentiated aggregates, we were able to detect the survival of rare TH  cells in two animals and a more numerous survival of TH  cells in a third (Figs. 8D, 8E). The data suggest that after an 8-week period, neural progenitors, but not large numbers of differentiated neurons, can survive and proliferate following implantation of the cell aggregates.
; U+ a3 o3 i  M* p7 g3 w: v4 V
Figure 8. Differentiation of cell clumps implanted into the lesioned rat brain. (A–C): Sections of implants from MedII/fibroblast growth factor 2–differentiated aggregates. (A): Hematoxylin and eosin staining of a section with a surviving implant. Putative neural precursor rosettes are indicated (arrows). (B): Implant detection using human-specific Alu probes and a human-specific nestin antibody. (C): Nestin expression in a large implant. (D): Chromogenic detection of surviving TH  cells derived from a DMEM/N2 transplant 8 weeks after implantation. The border of the graft is indicated (black dashed lines). (E): Higher magnification of the boxed region (white dashed) in (D). Scale bars = 250 μm (A), 100 μm (B, C, D), and 50 μm (E).Abbreviations: COR, cortex; DMEM, Dulbecco’s modified Eagle’s medium; STR, striatum; TH, tyrosine hydroxylase.

DISCUSSION

We have developed a simple culture system for the differentiation of hESCs to enriched neuronal populations of cells, including those of the midbrain dopaminergic lineage, characterized the expression of a variety of neuronal and dopaminergic markers, and demonstrated the functionality expected of differentiating neurons. This differentiation system could provide a simple experimental model for developing optimal cultures of midbrain dopaminergic populations suitable for implantation studies in animal models of PD and possible therapeutic applications.* s* H3 {4 l* U; T
7 b: S0 U# R4 t) B4 n8 Z" I
NOTE ADDED IN PROOF
$ t& e* e2 s( Y2 G# K/ f
We thank current and former members of the BresaGen Cell Therapy Programs in Adelaide and Athens, Ray Johnson for performing HPLC analysis, Clifton Baile and Diane Hartzel and the Animal Facility of the University of Georgia, Animal and Dairy Science Department for performing rat implantations, and Mahendra Rao for critically reading the manuscript. This work was supported by BresaGen Inc. and the Augusta Chapter of the American Legion (to N.A.L.). hESC characterization work was also supported by NIH grant R24DK063689 (to B.G.C., awarded to BresaGen Inc.).

REFERENCES3 Q1 `4 N& a+ i' \( y# ]
+ G; h1 M  Q: \* y7 V4 e  x
Freed CR. Will embryonic stem cells be a useful source of dopamine neurons for transplant into patients with Parkinson’s disease? Proc Natl Acad Sci U S A 2002;99:1755–1757.
* C4 M; x' l! D5 l  Z
Carpenter MK, Rosler ES, Fisk GJ et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 2004;229:243–258.- r: R; q; R- z6 Z- f# B8 ]
3 w1 p" b* b9 A- M6 |' V
Sato N, Meijer L, Skaltsounis L et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.7 \; w4 M3 @( k5 T" w3 |0 M

Rosler ES, Fisk GJ, Ares X et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 2004;229:259–274.& I6 F  f2 {# G) ^7 u) U

Bjorklund A, Dunnett SB, Brundin P et al. Neural transplantation for the treatment of Parkinson’s disease. Lancet Neurol 2003;2:437–445.6 \# C3 P4 M% V- i* D. i, T
+ {& v' _; v+ U, m
Freed CR, Greene PE, Breeze RE et al. Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N Engl J Med 2001;344:710–719.; |. @0 F2 Y$ B( v
& C; J. y8 ?0 m0 c" Q: ?
Olanow CW, Goetz CG, Kordower JH et al. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson’s disease. Ann Neurol 2003;54:403–414.
  v0 A: d! G3 }& S7 P$ k
Isacson O, Bjorklund LM, Schumacher JM. Toward full restoration of synaptic and terminal function of the dopaminergic system in Parkinson’s disease by stem cells. Ann Neurol 2003;53(suppl 3):S135–S146.2 B; X3 k9 [2 T) U

Barberi T, Klivenyi P, Calingasan NY et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 2003;21:1200–1207.9 O8 g: C$ r" A2 k0 ~+ W( A. q
) b% B  O' |; M& b  ~* i0 a
Bjorklund LM, Sanchez-Pernaute R, Chung S et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci U S A 2002;99:2344–2349.# h# Y( R+ n7 t  C  F+ m% ~

Kim JH, Auerbach JM, Rodriguez-Gomez JA et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 2002; 418:50–56.

Kawasaki H, Mizuseki K, Nishikawa S et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000;28:31–40.
( T8 [) P" P6 l1 H  V
Kawasaki H, Suemori H, Mizuseki K et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 2002;99:1580–1585.

Lee SH, Lumelsky N, Studer L et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000;18:675–679.6 z. {$ a9 R3 o

Schulz TC, Palmarini GM, Noggle SA et al. Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci 2003;4:27.: V/ w! E! q( Y4 p0 ]
8 T2 |, @2 _) X' t* A; n
Mitalipova MM, Calhoun JD, Shin S et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526.

Rathjen J, Lake JA, Bettess MD et al. Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J Cell Sci 1999;112:601–612.! s4 H  v3 g6 y# O( ?

Calhoun JD, Lambert NA, Mitalipova MM et al. Differentiation of rhesus embryonic stem cells to neural progenitors and neurons. Biochem Biophys Res Commun 2003;306: 191–197.# N2 E4 c0 K7 ]( C/ }! f$ U

Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002;30:e36.

Studer L, Tabar V, McKay RD. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1998;1:290–295.

Brustle O, Choudhary K, Karram K et al. Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat Biotechnol 1998;16: 1040–1044.
3 R4 I( p7 ?* t* ~. y, s
Buzzard JJ, Gough NM, Crook JM et al. Karyotype of human ES cells during extended culture. Nat Biotechnol 2004;22:381–382.8 c* o* Y- ]$ ^, k# ]5 z- [% ^, I

Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.% [' |% _. u6 U2 \: F

Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.

Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.

Sato N, Sanjuan IM, Heke M et al. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 2003;260:404–413.

Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004;269:360–380.

Bhattacharya B, Miura T, Brandenberger R et al. Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 2004;103:2956–2964.  y6 i  E: j( w+ T% Q
. V" s1 \+ J! N; K7 Y
Reubinoff BE, Itsykson P, Turetsky T et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19:1134–1140.
) S' L! t/ J3 f! j3 k% H3 e
Rathjen J, Haines BP, Hudson KM et al. Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population. Development 2002;129:2649–2661.1 o4 `4 w" [  G$ u* w% T; p3 l
6 I8 @9 }2 V! w. b) S5 R
Carpenter MK, Inokuma MS, Denham J et al. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 2001;172:383–397.% W- |) p3 w+ [$ H: m- |
* u6 m: Z2 h7 W" l
Zhang SC, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–1133.
: A* R' S. M, I4 R
Okano HJ, Darnell RB. A hierarchy of Hu RNA binding proteins in developing and adult neurons. J Neurosci 1997; 17:3024–3037.
  ~% D. x& G6 p4 T! j: ^, j
Mahadevan LC, Willis AC, Barratt MJ. Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors. Cell 1991;65:775–783.
/ a) S  F7 C, b2 ]3 x
Zeng X, Miura T, Lou Y et al. Properties of pluripotent human embryonic stem cells BG01 and BG02. STEM CELLS 2004;22:292–312.) G2 \: h! n# t  T
7 ~  ]  U# H( P) H) p  K0 m" a2 {# a6 h
Luo Y, Cai J, Ginis I et al. Designing, testing, and validating a focused stem cell microarray for characterization of neural stem cells and progenitor cells. STEM CELLS 2003;21:575–587.

Luo Y, Cai J, Liu Y et al. Microarray analysis of selected genes in neural stem and progenitor cells. J Neurochem 2002;83:1481–1497.

Wiles MV, Johansson BM. Embryonic stem cell development in a chemically defined medium. Exp Cell Res 1999;247:241–248.- M/ R; y" L2 ^

Finley MF, Devata S, Huettner JE. BMP-4 inhibits neural differentiation of murine embryonic stem cells. J Neurobiol 1999;40:271–287.6 c5 p0 L7 b  N$ t* a

Munoz-Sanjuan I, Brivanlou AH. Neural induction, the default model and embryonic stem cells. Nat Rev Neurosci 2002;3:271–280., p& p' q* s2 s( W
4 G3 M2 c$ N5 e+ z' z/ t
Sasai Y, De Robertis EM. Ectodermal patterning in vertebrate embryos. Dev Biol 1997;182:5–20.- b2 J8 m+ C- A0 d6 P' a0 R

Jessell TM. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet 2000;1:20–29.6 j0 Q7 Z2 Y5 O9 z. r# v/ o
$ }6 }: w: m4 t& x) b! a) H
Knecht AK, Bronner-Fraser M. Induction of the neural crest: a multigene process. Nat Rev Genet 2002;3:453–461.

Troy CM, Muma NA, Greene LA et al. Regulation of peripherin and neurofilament expression in regenerating rat motor neurons. Brain Res 1990;529:232–238.& S2 s# {/ O( i% ~

Mizuseki K, Sakamoto T, Watanabe K et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc Natl Acad Sci U S A 2003;100:5828–5833.
! Q! S( I! c+ X! F* r- Y  W
Smidt MP, Asbreuk CH, Cox JJ et al. A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b. Nat Neurosci 2000;3:337–341.( p" {3 I+ u2 V
+ l* y% e1 p, V! W2 j7 t; ?
Asbreuk CH, Vogelaar CF, Hellemons A et al. CNS expression pattern of Lmx1b and coexpression with ptx genes suggest functional cooperativity in the development of fore-brain motor control systems. Mol Cell Neurosci 2002;21: 410–420.
0 ]6 i8 [. w7 s; [1 m) C/ S
Bylund M, Andersson E, Novitch BG et al. Vertebrate neurogenesis is counteracted by Sox1-3 activity. Nat Neurosci 2003;6:1162–1168.5 L/ [; R6 j. [# G& f% M% c$ P1 Y- W

Pevny LH, Sockanathan S, Placzek M et al. A role for SOX1 in neural determination. Development 1998;125:1967–1978.( T6 d* [; m# g

Saucedo-Cardenas O, Quintana-Hau JD, Le WD et al. Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Natl Acad Sci U S A 1998;95:4013–4018.
4 o1 {% @  o0 A
Simon HH, Saueressig H, Wurst W et al. Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J Neurosci 2001;21:3126–3134.- r' ?% m1 c7 V1 T* h% `: ?
; @! A! {! f/ G
Zetterstrom RH, Solomin L, Jansson L et al. Dopamine neuron agenesis in Nurr1-deficient mice. Science 1997;276: 248–250.; u$ a; D/ n' |, f
$ I9 }2 r+ H# p! R# X" `3 x  f
Castillo SO, Baffi JS, Palkovits M et al. Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene. Mol Cell Neurosci 1998;11:36–46.% Q) k8 q+ o+ x& x, E* ?1 u; {
* h# e8 g' \. S: J% O* b% s
Nunes I, Tovmasian LT, Silva RM et al. Pitx3 is required for development of substantia nigra dopaminergic neurons. Proc Natl Acad Sci U S A 2003;100:4245–4250.9 I9 P' s6 g, D& B1 `
$ M- p- z" c% K7 z1 I9 Q! e2 ~
van den Munckhof P, Luk KC, Ste-Marie L et al. Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons. Development 2003;130: 2535–2542.$ p2 f8 K. d2 m" b# ]" W
, p: K9 m/ ]2 H6 w6 t
Hwang DY, Ardayfio P, Kang UJ et al. Selective loss of dopaminergic neurons in the substantia nigra of Pitx3-deficient aphakia mice. Brain Res Mol Brain Res 2003;114: 123–131.5 c; c# H. D' k4 {
0 k; S! }) u( o4 H( B- ^
Schein JC, Hunter DD, Roffler-Tarlov S. Girk2 expression in the ventral midbrain, cerebellum, and olfactory bulb and its relationship to the murine mutation weaver. Dev Biol 1998;204:432–450.$ D2 C" Q  Z$ e& O( L! Z2 ^
/ l8 F  i3 Y* ]7 X( W0 _3 N
Yamada T, McGeer PL, Baimbridge KG et al. Relative sparing in Parkinson’s disease of substantia nigra dopamine neurons containing calbindin-D28K. Brain Res 1990;526: 303–307.$ v; ]9 k6 G4 @6 j  O. K7 r

Gibb WR. Melanin, tyrosine hydroxylase, calbindin and substance P in the human midbrain and substantia nigra in relation to nigrostriatal projections and differential neuronal susceptibility in Parkinson’s disease. Brain Res 1992; 581:283–291.

German DC, Manaye K, Smith WK et al. Midbrain dopaminergic cell loss in Parkinson’s disease: computer visualization.Ann Neurol 1989;26:507–514.
2 M% X8 P# ~; [% E# ?
Rolletschek A, Chang H, Guan K et al. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors. Mech Dev 2001; 105:93–104.
" ~, y: _( w$ S% e8 _5 k& B
Lew JY, Garcia-Espana A, Lee KY et al. Increased site-specific phosphorylation of tyrosine hydroxylase accompanies stimulation of enzymatic activity induced by cessation of dopamine neuronal activity. Mol Pharmacol 1999;55:202–209.

Neve RL, Harris P, Kosik KS et al. Identification of cDNA clones for the human microtubule-associated protein tau and chromosomal localization of the genes for tau and microtubule-associated protein 2. Brain Res 1986;387: 271–280.
) u0 W+ {- O5 e
Amit M, Margulets V, Segev H et al. Human feeder layers for human embryonic stem cells. Biol Reprod 2003;68: 2150–2156.. w7 J0 C! `8 Z  V' `6 U/ o

Cheng L, Hammond H, Ye Z et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.) ?; H3 X# w4 k; e

Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002; 20:933–936." s" t$ B. s+ l
! \7 \/ R6 G  O! `6 l4 o7 U! G
Deumens R, Blokland A, Prickaerts J. Modeling Parkinson’s disease in rats: an evaluation of 6-OHDA lesions of the nigrostriatal pathway. Exp Neurol 2002;175:303–317.; L) P7 o/ U/ Z$ D$ I7 T+ T: ]
" `& F) ~8 p/ ~0 m
Studer L, Csete M, Lee SH et al. Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J Neurosci 2000;20:7377–7383.

Zeng X, Cai J, Chen J et al. Dopaminergic differentiation of human embryonic stem cells. STEM CELLS 2004;22:925–940.(Thomas C. Schulza, Scott )