标题: Generation of Tyrosine Hydroxylase Positive Neurons from Human Embryonic Stem Ce [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:36 标题: Generation of Tyrosine Hydroxylase Positive Neurons from Human Embryonic Stem Ce
Division of Clinical Pharmacology, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA7 j/ ]' p' N) N
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Key Words. Parkinson’s disease ? Astrocytes ? PA6 cells ? Dopamine neurons * p( J, Y! \& I% A N% X0 ^* s9 A A! l- o: hCorrespondence: Curt R. Freed, M.D., University of Colorado Health Sciences Center, 4200 East Ninth Avenue, C237, Denver, Colorado 80262, USA. Telephone: 303-315-8455; Fax: 303-315-3272; e-mail: Curt.Freed@UCHSC.edu ) N3 Z( e+ l& v/ _8 K / U0 h5 ?+ b$ k9 f* i6 A/ ^ABSTRACT l+ d. _; k6 A+ Q4 f c9 }) B
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Parkinson’s disease is characterized by the loss of midbrain neurons that synthesize dopamine. Pharmacological treatment with L-3,4-dihydroxyphenylalanine (L-dopa) works initially, but over time there is reduced efficacy along with motor complications . We and others have reported that transplantation of dopamine cells from the human embryonic mesencephalon, 7–8 weeks after conception, can improve motor function in people with advanced Parkinson’s disease . Limitations of this procedure include difficulty in obtaining embryonic brain tissue, as well as poor survival of the transplanted dopamine neurons. Human embryonic stem (hES) cells may provide an unlimited source of neural cells for transplantation if they can be differentiated into authentic dopamine neurons . % j4 w, Z; Q) Z2 o9 _8 a * S0 {7 a- c: H U8 Q) xES cells are isolated from the inner cell mass of the blastocyst . ES cells have the ability to remain undifferentiated and to proliferate indefinitely in vitro. When culture conditions are changed to allow for ES cell differentiation, derivatives of all three embryonic germ layers are produced . Transplantation of ES cells or their partially differentiated progeny, embryoid bodies, leads to the development of some dopamine neurons and teratomas . For hES cells to be used for transplantation into patients with Parkinson’s disease, they must be differentiated into dopamine neurons with no residual ES cells. The differentiation pattern of ES cells can be influenced by factors such as stromal cell–derived inducing activity (SDIA), which has been associated with PA6 cells . Coculturing PA6 cells with ES cells has resulted in the induction of neural progenitor cells and, subsequently, tyrosine hydroxylase (TH)–positive neurons in both mouse and nonhuman primate cells . We have sought to extend this work to human embryonic stem cells by differentiating them in cocultures with PA6 cells and astrocytes, as well as with the addition of glial-derived neurotrophic factor (GDNF).! M& Q; p9 @9 b1 \; B+ n- x3 D
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METHODS ( S5 ?4 X, _0 M4 P; y7 y2 y# `/ K# W' I/ }' g3 v1 c# F
In our earliest experiments with hES cells plated on PA6 cells, we observed relatively poor survival with an average of only one colony surviving out of 15 colonies plated. Nonetheless, we found that hES cells grown on PA6 cells could differentiate into TH-positive cells when they were cocultured for 4 weeks. Because the number of TH-positive cells was low under these conditions, we sought to enrich the differentiation environment by exposing the coculture to human embryonic striatal tissue. To prevent contamination of hES/PA6 cocultures with TH-positive cells from the striatum, the striatal cells were cultured in hanging baskets. This system permitted soluble factors released from the striatum to influence the hES cells. As shown in Figure 1, the combination of culture on PA6 cells plus soluble factors from human embryonic striatum led to differentiated colonies with a large TH-positive cell yield. * H8 f1 a9 ?. H2 w- F1 ~) t, G6 z0 ~) @' R I4 e/ T
Figure 1. Photomicrograph of a human embryonic stem cell colony plated on PA6 cells and exposed to embryonic human striatum for 4 weeks, then immunocytochemically stained for tyrosine hydroxylase (TH). This combined treatment produced a dense population of TH-positive cells (insets). Scale bar: 100 μm.! z) ^. ?8 }5 _/ q$ e
2 b+ B+ w. W4 m% c: e6 SFigure 2 presents quantitative results from cocultures of hES cells with PA6 cells and astrocytes. As shown in panel I, substrates and other treatments had a major effect on the number of TH-positive cells generated. In Experiment A, hES cells cocultured for 4 weeks on PA6 cells produced few TH-positive cells (13 ± 11 TH-positive cells per well). When cells were exposed to embryonic striatum in hanging baskets, cell number increased substantially (269 ± 89 TH-positive cells per well; p % @4 L5 ?# u. T9 q1 V. R
6 _* ]9 X6 Y$ S$ BFigure 2. Effects of differentiation conditions on (I) TH cell number, (II) colony number, and (III) colony circumference are shown after three separate experimental strategies. Experiment A: hES cells cultured for 4 weeks on PA6 cells (A1) or PA6 cells plus human embryonic striatum suspended in hanging baskets (A2). Experiment B: hES cells cultured for 3 weeks on rat embryonic mesencephalic astrocytes (B1) or on rat embryonic striatal astrocytes (B2). Experiment C: hES cells cultured for 3 weeks on a gelatin-coated substrate (C1), PA6 cells (C2), or PA6 cells with 10 ng/ml GDNF (C3). Using a gelatin-coated substrate produced no TH-positive cells. GDNF treatment at least doubled the TH-positive cell yield from differentiated hES cells (F = 22, p 7 z" h5 B o2 j6 ]/ E; R9 d: ]* A8 Y1 J
In Experiment C, we studied the effects of GDNF on the differentiation of TH-positive cells. Because GDNF is a factor produced by embryonic astrocytes and can promote survival and differentiation of dopamine neurons, we added GDNF to hES/PA6 cell cocultures and compared results with hES/PA6 alone or with hES cells on a gelatin-coated substrate after 3 weeks in culture. We found that GDNF doubled the number of TH-positive cells in cocultures with PA6 cells (PA6 GDNF: 934 ± 136; PA6 alone: 443 ± 105, or, on a gelatin-coated substrate: 0 ± 0 TH-positive cells per well). We also noted that the increase in TH-positive cell number was associated with greater colony survival. Of the 15 colonies placed in each well, PA6 GDNF led to survival of 13 ± 2 colonies per well, compared with 9 ± 1 colonies per well with PA6 alone or 2 ± 0 colonies per well for no cell substrate, p + G" \' Y# l" m R# ^( u" H n
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As shown in Figure 3, RT-PCR was performed to examine the effects of PA6 and GDNF on the transcription of genes linked to the dopaminergic neuronal phenotype. Results showed that gene transcription during differentiation on PA6 cells was increased by exposure to GDNF. Somewhat surprisingly, there was expression of engrailed-1, ptx3, and TH in the hES cells grown on a gelatin-coated plastic substrate, despite the fact that no TH-positive cells were seen. Because no TH protein was seen in cells differentiated without substrate, the PCR results show that while mRNA was generated, there was little or no protein translation. 1 U+ e6 A) {! s$ l* |" j $ C' D5 G. J7 u! w. D: b# KFigure 3. Reverse transcription polymerase chain reaction reveals the expression of several transcription factors involved in the development of dopamine neurons. The PA6 glial-derived neurotrophic factor group displayed an increased expression of all transcription factors measured. Tyrosine hydroxylase appears to be transcribed but not translated in the gelatin-coated substrate group. 9 I2 X7 r/ `; |" j : j/ e3 |3 C; _DISCUSSION : ]- d. ^4 ^& @; a K5 K6 V * G n, n0 A/ w. v/ FEmbryonic striatal astrocytes and PA6 stromal cells provide efficient substrates for differentiation of human embryonic stem cells into TH-positive neurons. The astrocyte-derived factor GDNF increased the overall number of TH-positive cells derived from human embryonic stem cells., q3 Z1 I/ C6 ` `
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