Dopaminergic Differentiation of Human Embryonic Stem Cells
http://www.100md.com
《干细胞学杂志》
a Cellular Neurobiology Branch, National Institute on Drug Abuse,
b Laboratory of Neuroscience, National Institute of Aging,
c Behavioral Neuroscience Branch, National Institute on Drug Abuse,
d Molecular Neuropsychiatry Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
Key Words. Embryonic stem cells ? Neuronal differentiation ? Dopaminergic PA6 stromal cells ? Hepatocyte growth factor
Correspondence: Xianmin Zeng, Ph.D., Development and Plasticity Section, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 333 Cassell Drive, Baltimore, Maryland 21224, USA. Telephone: 410-550-6565; Fax: 410-550-1621; e-mail: xzeng@intra.nida.nih.gov
ABSTRACT
Dopaminergic neurons have potential value in cell replacement therapy, especially for Parkinson’s disease , as well as for assessing the role of potential therapeutic agents in culture assays. A major constraint in the development of transplantation therapy for Parkinson’s disease has been the limited availability of human cells for both basic and therapeutic research. With respect to transplantation studies, the necessity of obtaining cells from individual fetuses results in logistical problems and difficulties in minimizing variability and optimizing cells for transplantation. At least as important as therapeutic transplantation, however, is the use of dopaminergic neurons for in vitro research on disease mechanisms, such as neuronal degeneration in Parkinson’s disease, and the role of dopaminergic neurons in the development and maintenance of drug abuse . If human dopaminergic neurons were readily available, studies on cellular mechanisms involved in these disorders could be greatly facilitated.
Human embryonic stem cells (hESCs), derived from the inner cell mass of preimplantation embryos, can proliferate indefinitely in culture and are able to differentiate into cell types of all three germ layers in vivo and in vitro . These unique properties of hESCs make them an excellent candidate as a source of functional differentiated cells for cell replacement therapies, provided that reliable means of inducing differentiation to specific cell types can be achieved. The first step to develop such cell-based therapeutics from hESCs is to define the appropriate conditions for the in vitro differentiation of hESCs into useful somatic cell types. Indeed, many clinically relevant cell types have been generated in vitro from mouse ES cells, and functional improvements have been achieved in rodent models after transplantation of dopaminergic neurons derived from mouse ES cells . Recently, differentiation of hESCs into multiple phenotypes, including neuronal , cardiomyogenic , hepatic , pancreatic , and hematopoietic lineages, has also been described.
Although several research groups have reported the generation of neural precursors and neurons from hESCs , none of these methods produces postmitotic mesencephalic dopamine neurons at a high frequency. On the other hand, dopaminergic neurons have been efficiently generated from mouse ES cells by two different methods. One is a multiple-step method involving in embryoid body (EB) formation followed by sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8) treatment and selection for nestin-positive cells . The second is a single-step method, which involves coculturing ES cells with a stromal cell line, PA6 . Of the two techniques, the system of coculturing with the PA6 cell line has the advantage of simplicity and speed and can be used with cells from subhuman primates . A simple and straightforward means of obtaining mature dopaminergic neurons from hESCs would be extremely valuable for both clinical application and for in vitro studies.
In this study we tested whether coculture with PA6 cells can induce differentiation of hESCs and found that a high frequency of dopaminergic neurons can be generated by this method. We show that the hES-derived cells generated appear to be authentic dopaminergic neurons of the mesencephalic type. These cells synthesize dopamine and release it under physiological stimuli, and these cells can be transplanted in a rat model of Parkinson’s disease.
MATERIALS AND METHODS
Neural Differentiation of hESCs Induced by PA6 Cells
The hESC line BG01 used in this study is strongly positive for several markers of undifferentiated ES cells, such as Oct4, SSEA-4, TRA-1-60, and TRA-1-81, but negative for NCAM (neural precursor and neuron marker, data not shown).
Neural differentiation of BG01 cells was initiated by culturing on a feeder layer of PA6 cells. By 6 days of culture on PA6 cells, most hES colonies had generated an outgrowth of elongated cells (Fig. 1A). By 10 days, extensive process formation was observed on the edges of most of the hESC colonies (Fig. 1B). After 2 weeks of differentiation, cells that migrated out of the colonies formed a monolayer and displayed a bipolar or multipolar morphology characteristic of neurons, with extensive development of fine processes (Fig. 1C). Prominent fiber bundles formed by processes emanating from the colonies were frequently observed.
Figure 1. Neural differentiation of hESCs induced by the stromal cell line PA6. (A–C): Phase-contrast photographs of hESCs during neural differentiation. (A):A colony with an outgrowth of elongated cells after 6 days of coculture with PA6 cells. (B): Neurite formation and typical bipolar cellular morphology at day 10. (C): By day 14, cells that migrated out of the colonies displayed bipolar or multipolar morphologies, with extensive development of fine neurites. (D–F): Immunostaining of hESC colonies after 12 days of culture on a layer of PA6 cells. (D): Nestin. (E): Neural cell adhesion molecule. (F): TuJ1. Antibody staining is in red (Nestin and TuJ1) or green (NCAM), whereas nuclear 4',6'-diamidino-2-phenylindole staining is in blue. (G): Time course of the appearance of neural markers during coculture of hESCs with PA6 cells. The percentage of colonies positive for each marker is shown as a function of the number of days cocultured with PA6 cells. NCAM first appeared on day 5, whereas TuJ1 appeared on day 8. TH-positive cells were first seen after 10 days. The scale bar is 20 μm. Abbreviations: hESC, human embryonic stem cell; NCAM, neural cell adhesion molecule; TH, tyrosine hydroxylase; TuJ1, neuron-specific class III beta tubulin.
At day 12, all hESC colonies were positive for the neural precursor marker nestin. Approximately 80% of hESC colonies contained NCAM-positive cells, and 70% of colonies were positive for the postmitotic neuronal marker TuJ1 (Figs. 1D–1F). Nestin was expressed during neural differentiation as well as in undifferentiated hESCs (data not shown). NCAM was first detected after 5 days of differentiation and increased for the next several days. NCAM peaked at day 14, at which time 92% of the colonies were NCAM positive (Fig. 1G). Likewise, colonies positive for TuJ1 first appeared at day 7, and the percentage of positive colonies increased during the following week of differentiation, so that by day 16, approximately 92% of the colonies were positive for TuJ1 (Fig. 1G). No Oct4- or SSEA-positive colonies were found after 3 weeks of differentiation on PA6 cells (data not shown).
hESCs Differentiate into Dopaminergic Neurons
We next tested whether TH-positive cells can be generated from hESCs by coculturing with PA6 cells. After 3 weeks of differentiation, approximately 87% of colonies contained TH-positive cells, and a high percentage of the cells in most of the colonies were TH positive (Figs. 2A–2C). TH-positive cells first appeared between 8 and 10 days after induction and peaked at approximately 18 days (Fig. 1G). Many cells were also positive for the synaptic proteins synapsin and synaptophysin (Figs. 2D–2E). Less than 10% of colonies contained GAD65-positive cells (not shown).
Figure 2. Dopaminergic differentiation of human embryonic stem cells. (A–C): Immunostaining of PA6-induced cells with a TH antibody after 18 days of coculture with PA6 cells. (A): A colony that contained TH-positive cells is shown. (B): A high percentage of the cells in the colonies was positive for TH. (C): Examples of individual cells that were positive for TH. (D, E): Immunostaining for the additional neuronal markers synapsin (D) and synaptophysin (E). (F): TH-positive cells (red) were negative for the noradrenergic marker DBH (green). (G): Double labeling with TH (green) and BrdU (red) antibodies after 24 hours of growth in the presence of BrdU. Numerous BrdU-positive cells were seen around the edges of the colony, but TH-positive cells were consistently BrdU negative, indicating that the TH-positive cells were postmitotic. Abbreviations: BrdU, bromodeoxyuridine; DBH, dopamine beta hydroxylase; TH, tyrosine hydroxylase.
More than 10 cultures were immunostained for DBH and were consistently negative. No DBH immunoreactivity was seen in TH-positive cells (Fig. 2F). BrdU incorporation for 24 hours showed that after 20 days of differentiation, most cells (72%) were not dividing. Double immunostaining of TH and BrdU showed no colocalization of TH and BrdU, indicating that the TH-positive cells were postmitotic (Fig. 2G). BrdU-positive cells were usually found outside colonies or around the edges of colonies, and very few BrdU-positive cells were present within colonies.
The expression of neuronal markers in PA6-induced cells was additionally analyzed by RT-PCR. Several dopaminergic markers, including TH, dopamine transporter (DAT), aromatic amino acid decarboxylase (AADC), and quinoid dihydropteridine reductase, were detected by RT-PCR (Fig. 3A). Receptors such as TrkB and TrkC, GFRA1, and the Shh-mediated protein smoothed (Smo) were also expressed in the PA6-induced cultures but not in undifferentiated hESCs. Several markers of non-dopaminergic neuronal sub-types were also present. The cholinergic markers choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), and the glutamatergic marker glutaminase (two isoforms, GAC and KGA) were detected in differentiated cells. DBH was not detected.
Figure 3. Expression of neuronal markers in hESC-derived neurons. (A): Reverse transcription–polymerase chain reaction analysis of markers of dopaminergic neurons, other neuronal markers, and stem cell markers using RNA from either undifferentiated hESCs or PA6-induced cells at day 21. (B): Quantification of Nurr1 mRNA in hESC-derived neurons and undifferentiated cells. Amplification curves corresponding to PCR4/Nr4a2 plasmid (standard curve) are shown from left to right, as follows: 18 pg/μl, 1.8 pg/μl, 0.18 pg/μl, and 0.018 pg/μl. Amplification curves corresponding to hESC-derived neurons (blue, red, and yellow curves) and undifferentiated cells (red, green, and blue curves) are represented by the first and second cluster of amplifications (from left to right; triplicates shown). Abbreviation: hESC, human embryonic stem cell.
The transcription factors that are known to control dopaminergic differentiation, Ptx3, Lmx1b, and Nurr1, and neurectodermal-specific transcription factor Sox1 were also detected (Fig. 3A). Most of the stem cell markers that were expressed in undifferentiated cells, including Oct4, hTERT, UTF-1, and Dppa5, were not detected or were downregulated in the differentiated cultures (Fig. 3A).
Because of the importance of Nurr1 in the generation of dopaminergic neurons, we performed quantitative RT-PCR to compare Nurr1 expression in PA6-induced neurons to undifferentiated hESCs. Nurr1 transcript was detected in both undifferentiated hESCs and PA6-induced differentiated cells, but expression of the Nurr1 transcript was threefold higher in the cultures differentiated for 3 weeks (Fig. 3B). Absolute numbers of copies of the Nurr1 transcript were 8 and 24 per 1,000 copies of the cyclophilin transcript for the undifferentiated cells and PA6-induced neurons, respectively.
Reverse-phase HPLC was used to examine the ability of hESC-derived neurons to release dopamine. After 3 weeks of differentiation, dopamine (2.8 ± 0.4 nM, from 107 to 108 cells) was released into the medium in response to a K+ depolarizing stimulus (Fig. 4), indicating that functional dopaminergic neurons were present in the culture. Noradrenalin (NA) was not detected in three of the four samples tested, although a small possible peak was seen in one sample, which is shown in Figure 4.
Figure 4. Dopamine release by K+ depolarization. A representative superimposed chromatogram showing the retention time of the dopamine peak for standards (red), a sample treated with 56 mM KCl for 15 minutes (blue), and a control sample without KCl treatment (green). In most cases, no NA peak was seen; the experiment shown is the only example for which a slight possible NA peak was seen. Dopamine was not detected in the supernatant before KCl treatment or in the medium before washing with Hanks’balanced salt solution. Abbreviations: DA, dopamine; DOPAC, 3,4-dihyroxphenylactic acid; HVA, homovanillic acid; NA, novadrenalin.
Gene Expression Profile of Differentiated Cells by a Human Stem Cell–Focused Array
A focused microarray containing cDNA probes for transcripts associated with different human stem cell populations was used to analyze gene expression in differentiated cultures compared with undifferentiated hESCs. Of the 266 genes represented by the array, 50 genes were expressed in the induced neurons but not detected in undifferentiated cells (Fig. 5, Table 2). These included 14 markers for stem and differentiated cells, 22 growth factors and receptors, adhesion molecules, and cytokines, six extracellular matrix molecules, and eight others (Fig. 5, Table 2). In particular, Sox1, Map2, TrkC, and NT3 were expressed at higher levels in the differentiated cultures, which is consistent with the results obtained by RT-PCR.
Figure 5. Gene expression profile of neurons derived from hESCs by a focused human stem cell microarray. Images of arrays hybridized using undifferentiated hESCs and cells that had been differentiated on PA6 cells. Abbreviations: hESC, human embryonic stem cells.
Table 2. Differentiation of BG01 cells on different feeder layers or growth factors
Differentiation Induced by PA6 Cells in the Presence of Growth Factors
In an attempt to identify the molecular nature of the neuron-inducing effect of PA6 cells, we used the MM-003N cytokine growth factor–focused array to compare PA6 cells with MEFs. In general, cytokine and growth factor expression patterns were remarkably similar for PA6 cells and MEFs. Hepatocyte growth factor (HGF) mRNA was, however, expressed at sixfold higher levels in PA6 cells than in MEFs (Fig. 6A). Greater expression of vascular endothelial growth factor (VEGF) and FGF7 mRNAs was also found in PA6 cells (Fig. 6A). RT-PCR confirmed that transcripts of HGF, VEGF, and FGF7 were increased in PA6 cells compared with MEFs (Figs. 6B, Table 3).
Figure 6. Gene expression profile for PA6 cells and MEFs by a focused murine cytokine microarray. (A): Images of arrays hybridized using RNA from either PA6 cells or MEFs. (B): Reverse transcription–PCR confirmation of differentially expressed genes. Abbreviations: MEF, mouse embryonic fibroblast; PCR, polymerase chain reaction.
Table 3. Mean number of cells per section positive for tyrosine hydroxylase (TH) or smooth muscle actin (SMA)
To determine whether these growth factors play a role in inducing neuronal differentiation, we compared differentiation of hESCs in the presence or absence of HGF,VEGF, and FGF7 using either PA6 cells or MEFs as feeder layers. No significant difference in the number of TH-positive colonies was observed among the cells differentiated either with or without HGF, VEGF, and FGF7 or with anti-HGF when cocultured with PA6 cells (data not shown). When hESCs were grown on PA6 cell membranes or lysed PA6 cells, with or without HGF, VEGF, and FGF7, few colonies survived and neuronal differentiation was not seen.
Because bone morphogenic protein (BMP) and serum have been demonstrated to inhibit neural differentiation in mouse and primate ES cells, we also tested whether BMP and serum have a similar effect on neural differentiation of hESCs induced by PA6 cells. When BMP4 or FCS was added to the medium, less than 10% of the colonies were TH-positive after 3 weeks of differentiation, indicating that BMP4 and serum suppressed neural differentiation of hESCs.
Transplantation of PA6-Induced Cells into the Rat Brain
To test whether PA6-induced dopaminergic cells derived from hESCs could be integrated in vivo, we transplanted these cells into the striatum of rats that had received unilateral lesions with 6-OHDA 4 weeks before transplantation and had been tested for amphetamine-induced rotation to verify lesion completeness. Two differentiation stages were used, early neural precursors after 8 days of coculture on PA6 cells and postmitotic neurons after 22 days of coculture on PA6 cells. Two animals received transplants of undifferentiated hESCs.
Immunohistochemical analysis was performed 5 weeks after grafting, and human cells were found in all brains transplanted with either 8- or 22-day cells. Some TH-positive human cells were also identified in both groups of animals (Figs. 7A–7E). Numbers of TH-positive cells were small, but a few clearly stained cells were found throughout the graft sites. In addition, larger numbers of weakly positive cells and cells with unclear morphology were also seen (Fig. 7F). An approximation of relative numbers of TH-positive cells was made by counting the total number of TH-positive cell profiles (clear cell morphology with unstained nucleus) for 10 sections from each animal (Table 3). Greater numbers of TH-positive cells were found in the brains of animals transplanted with 22-day differentiated cells compared with the animals transplanted with 8-day differentiated cells.
Figure 7. Immunohistochemistry of hESCs after transplantation into the brain of cyclosporin-immunosuppressed rats with 6-hydroxydopamine lesions. (A–E): TH-immunoreactive cells double stained for anti-human antigen. TH staining is in green, and anti-human antigen is in red. Nuclei are stained with DAPI (blue). (A, B): hESCs differentiated for 8 days before transplantation. (C–E): Cells differentiated for 22 days before transplantation. (F): Low magnification of a section from an animal transplanted with cells differentiated for 22 days, showing a larger number of TH-immunoreactive cells with unclear neuronal morphology. These cells, which are triple labeled with TH (green), anti-human antigen (red), and DAPI (blue), appear white. The red cells are transplanted hESCs, which are negative for TH. (G–I): Sections are immunostained for smooth muscle actin (red), a marker for a non–ventral nervous system cells. Nuclei are stained with DAPI (blue). (G): Section from an animal transplanted with undifferentiated cells. (H): Cells differentiated for 8 days before transplantation. (I): Cells differentiated for 22 days before transplantation. Abbreviations: DAPI, 4',6'-diamidino-2-phenylindole; hESC, human embryonic stem cell; TH, tyrosine hydroxylase.
No Oct4-positive cells were seen in any of the brains transplanted with either 8- or 22-day differentiated cells or undifferentiated cells, indicating that pluripotent hESCs did not persist or expand after transplantation. Smooth muscle actin (SMA) was chosen to monitor the presence of a cell type other than neuronal ectoderm derived from hESCs in the transplanted cells. SMA-positive cells were found in all of the animals. The number of SMA-positive cells in the animals transplanted with 22-day cells was approximately half that seen in animals transplanted with 8-day cells (Figs. 7G, 7H; Table 5). Larger numbers of SMA-positive cells were found in the animals that received transplants of undifferentiated cells (Fig. 7I; Table 5). Thus, coculture with PA6 cells did not preclude the differentiation of a significant number of cells with mesodermal properties.
DISCUSSION
Collier TJ, Steece-Collier K, McGuire S et al. Cellular models to study dopaminergic injury responses. Ann N Y Acad Sci 2003;991:140–151.
Lindvall O, Hagell P. Cell therapy and transplantation in Parkinson’s disease. Clin Chem Lab Med 2001;39:356–361.
Perrier AL, Studer L. Making and repairing the mammalian brain: in vitro production of dopaminergic neurons. Semin Cell Dev Biol 2003;14:181–189.
Jentsch JD, Roth RH, Taylor JR. Role for dopamine in the behavioral functions of the prefrontal corticostriatal system: implications for mental disorders and psychotropic drug action. Prog Brain Res 2000;126:433–453.
Pierce RC, Bari AA. The role of neurotrophic factors in psychostimulant-induced behavioral and neuronal plasticity. Rev Neurosci 2001;12:95–110.
Volkow ND, Fowler JS, Wang GJ et al. Role of dopamine in the therapeutic and reinforcing effects of methylphenidate in humans: results from imaging studies. Eur Neuropsychopharmacol 2002;12:557–566.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
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.
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.
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.
Reubinoff BE, Itsykson P, Turetsky T et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19:1134–1140.
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.
Kehat I, Gepstein A, Spira A et al. High-resolution electro-physiological assessment of human embryonic stem cell-derived cardiomyocytes: a novel in vitro model for the study of conduction. Circ Res 2002;91:659–661.
Xu C, Police S, Rao N et al. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 2002;91:501–508.
Rambhatla L, Chiu CP, Kundu P et al. Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant 2003;12:1–11.
Assady S, Maor G, Amit M et al. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–1697.
Kaufman DS, Hanson ET, Lewis RL et al. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2001;98:10716–10721.
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.
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.
Zeng X, Miura T, Luo Y et al. Properties of pluripotent human embryonic stem cells BG01 and BG02. STEM CELLS 2004;22:292–312.
Zeng X, Chen J, Sanchez JF et al. Stable expression of hrGFP by mouse embryonic stem cells: promoter activity in the undifferentiated state and during dopaminergic neural differentiation. STEM CELLS 2003;21:647–653.
Luo Y, Cai J, Ginis I et al. Designing, testing and validating a stem cell microarray for characterization of neural stem cells and progenitor cells. STEM CELLS 2003;21:575–587.
Wang Y, Lin JC, Chiou AL et al. Human ventromesencephalic grafts restore dopamine release and clearance in hemiparkinsonian rats. Exp Neurol 1995;136:98–106.
Park S, Lee KS, Lee YJ et al. Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors. Neurosci Lett 2004;359:99–103.
Park S, Kim EY, Ghil GS et al. Genetically modified human embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson’s disease. Neurosci Lett 2003;353:91–94.
Lindvall O. Stem cells for cell therapy in Parkinson’s disease. Pharmacol Res 2003;47:279–287.
Marchionini DM, Collier TJ, Camargo M et al. Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson’s disease. J Comp Neurol 2003;464:172–179.
Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003;425:968–973.
Lindvall O, Bjorklund A. Anatomy of the dopaminergic neuron systems in the rat brain. Adv Biochem Psychopharmacol 1978;19:1–23.
Simon HH, Bhatt L, Gherbassi D et al. Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors. Ann N Y Acad Sci 2003;991:36–47.
Liu J, Merlie JP, Todd RD et al. Identification of cell type-specific promoter elements associated with the rat tyrosine hydroxylase gene using transgenic founder analysis. Brain Res Mol Brain Res 1997;50:33–42.
Sakurada K, Ohshima-Sakurada M, Palmer TD et al. Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain. Development 1999;126:4017–4026.
Ye W, Shimamura K, Rubenstein JL et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998;93:755–766.
Pevny LH, Sockanathan S, Placzek M et al. A role for SOX1 in neural determination. Development 1998;125:1967–1978.
Zetterstrom RH, Solomin L, Jansson L et al. Dopamine neuron agenesis in Nurr1-deficient mice. Science 1997;276:248–250.
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.
Wang MZ, Jin P, Bumcrot DA, Marigo V et al. Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein. Nat Med 1995;1:1184–1188.
Ericson J, Muhr J, Placzek M et al. Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 1995;81:747–756.
Murone M, Rosenthal A, de Sauvage FJ. Sonic hedgehog signaling by the patched-smoothened receptor complex. Curr Biol 1999;9:76–84.
Marigo V, Davey RA, Zuo Y et al. Biochemical evidence that patched is the Hedgehog receptor. Nature 1996;384:176–179.
Stone DM, Hynes M, Armanini M et al. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 1996;384:129–134.
MacArthur CA, Lawshe A, Xu J et al. FGF-8 isoforms activate receptor splice forms that are expressed in mesenchymal regions of mouse development. Development 1995; 121:3603–3613.
Hyman C, Hofer M, Barde YA et al. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 1991;350:230–232.
Knusel B, Winslow JW, Rosenthal A et al. Promotion of central cholinergic and dopaminergic neuron differentiation by brain-derived neurotrophic factor but not neurotrophin 3. Proc Natl Acad Sci U S A 1991;88:961–965.
Lin LF, Doherty DH, Lile JD et al. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 1993;260:1130–1132.
Erickson JT, Brosenitsch TA, Katz DM. Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor are required simultaneously for survival of dopaminergic primary sensory neurons in vivo. J Neurosci 2001;21:581–589.
Soppet D, Escandon E, Maragos J et al. The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor. Cell 1991;65:895–903.
Klein R, Nanduri V, Jing SA et al. The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 1991;66:395–403.
Nosrat CA, Tomac A, Hoffer BJ et al. Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs. Exp Brain Res 1997;115:410–422.
Takahashi M. The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev 2001;12:361–373.
Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004;269:360–380.
Hamanoue M, Takemoto N, Matsumoto K et al. Neurotrophic effect of hepatocyte growth factor on central nervous system neurons in vitro. J Neurosci Res 1996;43:554–564.(Xianmin Zenga, Jingli Cai)
b Laboratory of Neuroscience, National Institute of Aging,
c Behavioral Neuroscience Branch, National Institute on Drug Abuse,
d Molecular Neuropsychiatry Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
Key Words. Embryonic stem cells ? Neuronal differentiation ? Dopaminergic PA6 stromal cells ? Hepatocyte growth factor
Correspondence: Xianmin Zeng, Ph.D., Development and Plasticity Section, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 333 Cassell Drive, Baltimore, Maryland 21224, USA. Telephone: 410-550-6565; Fax: 410-550-1621; e-mail: xzeng@intra.nida.nih.gov
ABSTRACT
Dopaminergic neurons have potential value in cell replacement therapy, especially for Parkinson’s disease , as well as for assessing the role of potential therapeutic agents in culture assays. A major constraint in the development of transplantation therapy for Parkinson’s disease has been the limited availability of human cells for both basic and therapeutic research. With respect to transplantation studies, the necessity of obtaining cells from individual fetuses results in logistical problems and difficulties in minimizing variability and optimizing cells for transplantation. At least as important as therapeutic transplantation, however, is the use of dopaminergic neurons for in vitro research on disease mechanisms, such as neuronal degeneration in Parkinson’s disease, and the role of dopaminergic neurons in the development and maintenance of drug abuse . If human dopaminergic neurons were readily available, studies on cellular mechanisms involved in these disorders could be greatly facilitated.
Human embryonic stem cells (hESCs), derived from the inner cell mass of preimplantation embryos, can proliferate indefinitely in culture and are able to differentiate into cell types of all three germ layers in vivo and in vitro . These unique properties of hESCs make them an excellent candidate as a source of functional differentiated cells for cell replacement therapies, provided that reliable means of inducing differentiation to specific cell types can be achieved. The first step to develop such cell-based therapeutics from hESCs is to define the appropriate conditions for the in vitro differentiation of hESCs into useful somatic cell types. Indeed, many clinically relevant cell types have been generated in vitro from mouse ES cells, and functional improvements have been achieved in rodent models after transplantation of dopaminergic neurons derived from mouse ES cells . Recently, differentiation of hESCs into multiple phenotypes, including neuronal , cardiomyogenic , hepatic , pancreatic , and hematopoietic lineages, has also been described.
Although several research groups have reported the generation of neural precursors and neurons from hESCs , none of these methods produces postmitotic mesencephalic dopamine neurons at a high frequency. On the other hand, dopaminergic neurons have been efficiently generated from mouse ES cells by two different methods. One is a multiple-step method involving in embryoid body (EB) formation followed by sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8) treatment and selection for nestin-positive cells . The second is a single-step method, which involves coculturing ES cells with a stromal cell line, PA6 . Of the two techniques, the system of coculturing with the PA6 cell line has the advantage of simplicity and speed and can be used with cells from subhuman primates . A simple and straightforward means of obtaining mature dopaminergic neurons from hESCs would be extremely valuable for both clinical application and for in vitro studies.
In this study we tested whether coculture with PA6 cells can induce differentiation of hESCs and found that a high frequency of dopaminergic neurons can be generated by this method. We show that the hES-derived cells generated appear to be authentic dopaminergic neurons of the mesencephalic type. These cells synthesize dopamine and release it under physiological stimuli, and these cells can be transplanted in a rat model of Parkinson’s disease.
MATERIALS AND METHODS
Neural Differentiation of hESCs Induced by PA6 Cells
The hESC line BG01 used in this study is strongly positive for several markers of undifferentiated ES cells, such as Oct4, SSEA-4, TRA-1-60, and TRA-1-81, but negative for NCAM (neural precursor and neuron marker, data not shown).
Neural differentiation of BG01 cells was initiated by culturing on a feeder layer of PA6 cells. By 6 days of culture on PA6 cells, most hES colonies had generated an outgrowth of elongated cells (Fig. 1A). By 10 days, extensive process formation was observed on the edges of most of the hESC colonies (Fig. 1B). After 2 weeks of differentiation, cells that migrated out of the colonies formed a monolayer and displayed a bipolar or multipolar morphology characteristic of neurons, with extensive development of fine processes (Fig. 1C). Prominent fiber bundles formed by processes emanating from the colonies were frequently observed.
Figure 1. Neural differentiation of hESCs induced by the stromal cell line PA6. (A–C): Phase-contrast photographs of hESCs during neural differentiation. (A):A colony with an outgrowth of elongated cells after 6 days of coculture with PA6 cells. (B): Neurite formation and typical bipolar cellular morphology at day 10. (C): By day 14, cells that migrated out of the colonies displayed bipolar or multipolar morphologies, with extensive development of fine neurites. (D–F): Immunostaining of hESC colonies after 12 days of culture on a layer of PA6 cells. (D): Nestin. (E): Neural cell adhesion molecule. (F): TuJ1. Antibody staining is in red (Nestin and TuJ1) or green (NCAM), whereas nuclear 4',6'-diamidino-2-phenylindole staining is in blue. (G): Time course of the appearance of neural markers during coculture of hESCs with PA6 cells. The percentage of colonies positive for each marker is shown as a function of the number of days cocultured with PA6 cells. NCAM first appeared on day 5, whereas TuJ1 appeared on day 8. TH-positive cells were first seen after 10 days. The scale bar is 20 μm. Abbreviations: hESC, human embryonic stem cell; NCAM, neural cell adhesion molecule; TH, tyrosine hydroxylase; TuJ1, neuron-specific class III beta tubulin.
At day 12, all hESC colonies were positive for the neural precursor marker nestin. Approximately 80% of hESC colonies contained NCAM-positive cells, and 70% of colonies were positive for the postmitotic neuronal marker TuJ1 (Figs. 1D–1F). Nestin was expressed during neural differentiation as well as in undifferentiated hESCs (data not shown). NCAM was first detected after 5 days of differentiation and increased for the next several days. NCAM peaked at day 14, at which time 92% of the colonies were NCAM positive (Fig. 1G). Likewise, colonies positive for TuJ1 first appeared at day 7, and the percentage of positive colonies increased during the following week of differentiation, so that by day 16, approximately 92% of the colonies were positive for TuJ1 (Fig. 1G). No Oct4- or SSEA-positive colonies were found after 3 weeks of differentiation on PA6 cells (data not shown).
hESCs Differentiate into Dopaminergic Neurons
We next tested whether TH-positive cells can be generated from hESCs by coculturing with PA6 cells. After 3 weeks of differentiation, approximately 87% of colonies contained TH-positive cells, and a high percentage of the cells in most of the colonies were TH positive (Figs. 2A–2C). TH-positive cells first appeared between 8 and 10 days after induction and peaked at approximately 18 days (Fig. 1G). Many cells were also positive for the synaptic proteins synapsin and synaptophysin (Figs. 2D–2E). Less than 10% of colonies contained GAD65-positive cells (not shown).
Figure 2. Dopaminergic differentiation of human embryonic stem cells. (A–C): Immunostaining of PA6-induced cells with a TH antibody after 18 days of coculture with PA6 cells. (A): A colony that contained TH-positive cells is shown. (B): A high percentage of the cells in the colonies was positive for TH. (C): Examples of individual cells that were positive for TH. (D, E): Immunostaining for the additional neuronal markers synapsin (D) and synaptophysin (E). (F): TH-positive cells (red) were negative for the noradrenergic marker DBH (green). (G): Double labeling with TH (green) and BrdU (red) antibodies after 24 hours of growth in the presence of BrdU. Numerous BrdU-positive cells were seen around the edges of the colony, but TH-positive cells were consistently BrdU negative, indicating that the TH-positive cells were postmitotic. Abbreviations: BrdU, bromodeoxyuridine; DBH, dopamine beta hydroxylase; TH, tyrosine hydroxylase.
More than 10 cultures were immunostained for DBH and were consistently negative. No DBH immunoreactivity was seen in TH-positive cells (Fig. 2F). BrdU incorporation for 24 hours showed that after 20 days of differentiation, most cells (72%) were not dividing. Double immunostaining of TH and BrdU showed no colocalization of TH and BrdU, indicating that the TH-positive cells were postmitotic (Fig. 2G). BrdU-positive cells were usually found outside colonies or around the edges of colonies, and very few BrdU-positive cells were present within colonies.
The expression of neuronal markers in PA6-induced cells was additionally analyzed by RT-PCR. Several dopaminergic markers, including TH, dopamine transporter (DAT), aromatic amino acid decarboxylase (AADC), and quinoid dihydropteridine reductase, were detected by RT-PCR (Fig. 3A). Receptors such as TrkB and TrkC, GFRA1, and the Shh-mediated protein smoothed (Smo) were also expressed in the PA6-induced cultures but not in undifferentiated hESCs. Several markers of non-dopaminergic neuronal sub-types were also present. The cholinergic markers choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), and the glutamatergic marker glutaminase (two isoforms, GAC and KGA) were detected in differentiated cells. DBH was not detected.
Figure 3. Expression of neuronal markers in hESC-derived neurons. (A): Reverse transcription–polymerase chain reaction analysis of markers of dopaminergic neurons, other neuronal markers, and stem cell markers using RNA from either undifferentiated hESCs or PA6-induced cells at day 21. (B): Quantification of Nurr1 mRNA in hESC-derived neurons and undifferentiated cells. Amplification curves corresponding to PCR4/Nr4a2 plasmid (standard curve) are shown from left to right, as follows: 18 pg/μl, 1.8 pg/μl, 0.18 pg/μl, and 0.018 pg/μl. Amplification curves corresponding to hESC-derived neurons (blue, red, and yellow curves) and undifferentiated cells (red, green, and blue curves) are represented by the first and second cluster of amplifications (from left to right; triplicates shown). Abbreviation: hESC, human embryonic stem cell.
The transcription factors that are known to control dopaminergic differentiation, Ptx3, Lmx1b, and Nurr1, and neurectodermal-specific transcription factor Sox1 were also detected (Fig. 3A). Most of the stem cell markers that were expressed in undifferentiated cells, including Oct4, hTERT, UTF-1, and Dppa5, were not detected or were downregulated in the differentiated cultures (Fig. 3A).
Because of the importance of Nurr1 in the generation of dopaminergic neurons, we performed quantitative RT-PCR to compare Nurr1 expression in PA6-induced neurons to undifferentiated hESCs. Nurr1 transcript was detected in both undifferentiated hESCs and PA6-induced differentiated cells, but expression of the Nurr1 transcript was threefold higher in the cultures differentiated for 3 weeks (Fig. 3B). Absolute numbers of copies of the Nurr1 transcript were 8 and 24 per 1,000 copies of the cyclophilin transcript for the undifferentiated cells and PA6-induced neurons, respectively.
Reverse-phase HPLC was used to examine the ability of hESC-derived neurons to release dopamine. After 3 weeks of differentiation, dopamine (2.8 ± 0.4 nM, from 107 to 108 cells) was released into the medium in response to a K+ depolarizing stimulus (Fig. 4), indicating that functional dopaminergic neurons were present in the culture. Noradrenalin (NA) was not detected in three of the four samples tested, although a small possible peak was seen in one sample, which is shown in Figure 4.
Figure 4. Dopamine release by K+ depolarization. A representative superimposed chromatogram showing the retention time of the dopamine peak for standards (red), a sample treated with 56 mM KCl for 15 minutes (blue), and a control sample without KCl treatment (green). In most cases, no NA peak was seen; the experiment shown is the only example for which a slight possible NA peak was seen. Dopamine was not detected in the supernatant before KCl treatment or in the medium before washing with Hanks’balanced salt solution. Abbreviations: DA, dopamine; DOPAC, 3,4-dihyroxphenylactic acid; HVA, homovanillic acid; NA, novadrenalin.
Gene Expression Profile of Differentiated Cells by a Human Stem Cell–Focused Array
A focused microarray containing cDNA probes for transcripts associated with different human stem cell populations was used to analyze gene expression in differentiated cultures compared with undifferentiated hESCs. Of the 266 genes represented by the array, 50 genes were expressed in the induced neurons but not detected in undifferentiated cells (Fig. 5, Table 2). These included 14 markers for stem and differentiated cells, 22 growth factors and receptors, adhesion molecules, and cytokines, six extracellular matrix molecules, and eight others (Fig. 5, Table 2). In particular, Sox1, Map2, TrkC, and NT3 were expressed at higher levels in the differentiated cultures, which is consistent with the results obtained by RT-PCR.
Figure 5. Gene expression profile of neurons derived from hESCs by a focused human stem cell microarray. Images of arrays hybridized using undifferentiated hESCs and cells that had been differentiated on PA6 cells. Abbreviations: hESC, human embryonic stem cells.
Table 2. Differentiation of BG01 cells on different feeder layers or growth factors
Differentiation Induced by PA6 Cells in the Presence of Growth Factors
In an attempt to identify the molecular nature of the neuron-inducing effect of PA6 cells, we used the MM-003N cytokine growth factor–focused array to compare PA6 cells with MEFs. In general, cytokine and growth factor expression patterns were remarkably similar for PA6 cells and MEFs. Hepatocyte growth factor (HGF) mRNA was, however, expressed at sixfold higher levels in PA6 cells than in MEFs (Fig. 6A). Greater expression of vascular endothelial growth factor (VEGF) and FGF7 mRNAs was also found in PA6 cells (Fig. 6A). RT-PCR confirmed that transcripts of HGF, VEGF, and FGF7 were increased in PA6 cells compared with MEFs (Figs. 6B, Table 3).
Figure 6. Gene expression profile for PA6 cells and MEFs by a focused murine cytokine microarray. (A): Images of arrays hybridized using RNA from either PA6 cells or MEFs. (B): Reverse transcription–PCR confirmation of differentially expressed genes. Abbreviations: MEF, mouse embryonic fibroblast; PCR, polymerase chain reaction.
Table 3. Mean number of cells per section positive for tyrosine hydroxylase (TH) or smooth muscle actin (SMA)
To determine whether these growth factors play a role in inducing neuronal differentiation, we compared differentiation of hESCs in the presence or absence of HGF,VEGF, and FGF7 using either PA6 cells or MEFs as feeder layers. No significant difference in the number of TH-positive colonies was observed among the cells differentiated either with or without HGF, VEGF, and FGF7 or with anti-HGF when cocultured with PA6 cells (data not shown). When hESCs were grown on PA6 cell membranes or lysed PA6 cells, with or without HGF, VEGF, and FGF7, few colonies survived and neuronal differentiation was not seen.
Because bone morphogenic protein (BMP) and serum have been demonstrated to inhibit neural differentiation in mouse and primate ES cells, we also tested whether BMP and serum have a similar effect on neural differentiation of hESCs induced by PA6 cells. When BMP4 or FCS was added to the medium, less than 10% of the colonies were TH-positive after 3 weeks of differentiation, indicating that BMP4 and serum suppressed neural differentiation of hESCs.
Transplantation of PA6-Induced Cells into the Rat Brain
To test whether PA6-induced dopaminergic cells derived from hESCs could be integrated in vivo, we transplanted these cells into the striatum of rats that had received unilateral lesions with 6-OHDA 4 weeks before transplantation and had been tested for amphetamine-induced rotation to verify lesion completeness. Two differentiation stages were used, early neural precursors after 8 days of coculture on PA6 cells and postmitotic neurons after 22 days of coculture on PA6 cells. Two animals received transplants of undifferentiated hESCs.
Immunohistochemical analysis was performed 5 weeks after grafting, and human cells were found in all brains transplanted with either 8- or 22-day cells. Some TH-positive human cells were also identified in both groups of animals (Figs. 7A–7E). Numbers of TH-positive cells were small, but a few clearly stained cells were found throughout the graft sites. In addition, larger numbers of weakly positive cells and cells with unclear morphology were also seen (Fig. 7F). An approximation of relative numbers of TH-positive cells was made by counting the total number of TH-positive cell profiles (clear cell morphology with unstained nucleus) for 10 sections from each animal (Table 3). Greater numbers of TH-positive cells were found in the brains of animals transplanted with 22-day differentiated cells compared with the animals transplanted with 8-day differentiated cells.
Figure 7. Immunohistochemistry of hESCs after transplantation into the brain of cyclosporin-immunosuppressed rats with 6-hydroxydopamine lesions. (A–E): TH-immunoreactive cells double stained for anti-human antigen. TH staining is in green, and anti-human antigen is in red. Nuclei are stained with DAPI (blue). (A, B): hESCs differentiated for 8 days before transplantation. (C–E): Cells differentiated for 22 days before transplantation. (F): Low magnification of a section from an animal transplanted with cells differentiated for 22 days, showing a larger number of TH-immunoreactive cells with unclear neuronal morphology. These cells, which are triple labeled with TH (green), anti-human antigen (red), and DAPI (blue), appear white. The red cells are transplanted hESCs, which are negative for TH. (G–I): Sections are immunostained for smooth muscle actin (red), a marker for a non–ventral nervous system cells. Nuclei are stained with DAPI (blue). (G): Section from an animal transplanted with undifferentiated cells. (H): Cells differentiated for 8 days before transplantation. (I): Cells differentiated for 22 days before transplantation. Abbreviations: DAPI, 4',6'-diamidino-2-phenylindole; hESC, human embryonic stem cell; TH, tyrosine hydroxylase.
No Oct4-positive cells were seen in any of the brains transplanted with either 8- or 22-day differentiated cells or undifferentiated cells, indicating that pluripotent hESCs did not persist or expand after transplantation. Smooth muscle actin (SMA) was chosen to monitor the presence of a cell type other than neuronal ectoderm derived from hESCs in the transplanted cells. SMA-positive cells were found in all of the animals. The number of SMA-positive cells in the animals transplanted with 22-day cells was approximately half that seen in animals transplanted with 8-day cells (Figs. 7G, 7H; Table 5). Larger numbers of SMA-positive cells were found in the animals that received transplants of undifferentiated cells (Fig. 7I; Table 5). Thus, coculture with PA6 cells did not preclude the differentiation of a significant number of cells with mesodermal properties.
DISCUSSION
Collier TJ, Steece-Collier K, McGuire S et al. Cellular models to study dopaminergic injury responses. Ann N Y Acad Sci 2003;991:140–151.
Lindvall O, Hagell P. Cell therapy and transplantation in Parkinson’s disease. Clin Chem Lab Med 2001;39:356–361.
Perrier AL, Studer L. Making and repairing the mammalian brain: in vitro production of dopaminergic neurons. Semin Cell Dev Biol 2003;14:181–189.
Jentsch JD, Roth RH, Taylor JR. Role for dopamine in the behavioral functions of the prefrontal corticostriatal system: implications for mental disorders and psychotropic drug action. Prog Brain Res 2000;126:433–453.
Pierce RC, Bari AA. The role of neurotrophic factors in psychostimulant-induced behavioral and neuronal plasticity. Rev Neurosci 2001;12:95–110.
Volkow ND, Fowler JS, Wang GJ et al. Role of dopamine in the therapeutic and reinforcing effects of methylphenidate in humans: results from imaging studies. Eur Neuropsychopharmacol 2002;12:557–566.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
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.
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.
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.
Reubinoff BE, Itsykson P, Turetsky T et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19:1134–1140.
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.
Kehat I, Gepstein A, Spira A et al. High-resolution electro-physiological assessment of human embryonic stem cell-derived cardiomyocytes: a novel in vitro model for the study of conduction. Circ Res 2002;91:659–661.
Xu C, Police S, Rao N et al. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 2002;91:501–508.
Rambhatla L, Chiu CP, Kundu P et al. Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant 2003;12:1–11.
Assady S, Maor G, Amit M et al. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691–1697.
Kaufman DS, Hanson ET, Lewis RL et al. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2001;98:10716–10721.
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.
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.
Zeng X, Miura T, Luo Y et al. Properties of pluripotent human embryonic stem cells BG01 and BG02. STEM CELLS 2004;22:292–312.
Zeng X, Chen J, Sanchez JF et al. Stable expression of hrGFP by mouse embryonic stem cells: promoter activity in the undifferentiated state and during dopaminergic neural differentiation. STEM CELLS 2003;21:647–653.
Luo Y, Cai J, Ginis I et al. Designing, testing and validating a stem cell microarray for characterization of neural stem cells and progenitor cells. STEM CELLS 2003;21:575–587.
Wang Y, Lin JC, Chiou AL et al. Human ventromesencephalic grafts restore dopamine release and clearance in hemiparkinsonian rats. Exp Neurol 1995;136:98–106.
Park S, Lee KS, Lee YJ et al. Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors. Neurosci Lett 2004;359:99–103.
Park S, Kim EY, Ghil GS et al. Genetically modified human embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson’s disease. Neurosci Lett 2003;353:91–94.
Lindvall O. Stem cells for cell therapy in Parkinson’s disease. Pharmacol Res 2003;47:279–287.
Marchionini DM, Collier TJ, Camargo M et al. Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson’s disease. J Comp Neurol 2003;464:172–179.
Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003;425:968–973.
Lindvall O, Bjorklund A. Anatomy of the dopaminergic neuron systems in the rat brain. Adv Biochem Psychopharmacol 1978;19:1–23.
Simon HH, Bhatt L, Gherbassi D et al. Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors. Ann N Y Acad Sci 2003;991:36–47.
Liu J, Merlie JP, Todd RD et al. Identification of cell type-specific promoter elements associated with the rat tyrosine hydroxylase gene using transgenic founder analysis. Brain Res Mol Brain Res 1997;50:33–42.
Sakurada K, Ohshima-Sakurada M, Palmer TD et al. Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain. Development 1999;126:4017–4026.
Ye W, Shimamura K, Rubenstein JL et al. FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate. Cell 1998;93:755–766.
Pevny LH, Sockanathan S, Placzek M et al. A role for SOX1 in neural determination. Development 1998;125:1967–1978.
Zetterstrom RH, Solomin L, Jansson L et al. Dopamine neuron agenesis in Nurr1-deficient mice. Science 1997;276:248–250.
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.
Wang MZ, Jin P, Bumcrot DA, Marigo V et al. Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein. Nat Med 1995;1:1184–1188.
Ericson J, Muhr J, Placzek M et al. Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 1995;81:747–756.
Murone M, Rosenthal A, de Sauvage FJ. Sonic hedgehog signaling by the patched-smoothened receptor complex. Curr Biol 1999;9:76–84.
Marigo V, Davey RA, Zuo Y et al. Biochemical evidence that patched is the Hedgehog receptor. Nature 1996;384:176–179.
Stone DM, Hynes M, Armanini M et al. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 1996;384:129–134.
MacArthur CA, Lawshe A, Xu J et al. FGF-8 isoforms activate receptor splice forms that are expressed in mesenchymal regions of mouse development. Development 1995; 121:3603–3613.
Hyman C, Hofer M, Barde YA et al. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 1991;350:230–232.
Knusel B, Winslow JW, Rosenthal A et al. Promotion of central cholinergic and dopaminergic neuron differentiation by brain-derived neurotrophic factor but not neurotrophin 3. Proc Natl Acad Sci U S A 1991;88:961–965.
Lin LF, Doherty DH, Lile JD et al. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 1993;260:1130–1132.
Erickson JT, Brosenitsch TA, Katz DM. Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor are required simultaneously for survival of dopaminergic primary sensory neurons in vivo. J Neurosci 2001;21:581–589.
Soppet D, Escandon E, Maragos J et al. The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor. Cell 1991;65:895–903.
Klein R, Nanduri V, Jing SA et al. The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 1991;66:395–403.
Nosrat CA, Tomac A, Hoffer BJ et al. Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs. Exp Brain Res 1997;115:410–422.
Takahashi M. The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev 2001;12:361–373.
Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004;269:360–380.
Hamanoue M, Takemoto N, Matsumoto K et al. Neurotrophic effect of hepatocyte growth factor on central nervous system neurons in vitro. J Neurosci Res 1996;43:554–564.(Xianmin Zenga, Jingli Cai)