摘要
在這項研究中，我們著重調查從人類胚胎幹細胞到多巴胺神經元的顯著功能模塊和信號通路的分化過程。藉由數據整合和資料庫探勘，我們對擴展和分化的人類胚胎幹細胞過程，建構一個候選的蛋白質網絡。然後，在基於三個胚胎幹細胞分化的條件下的基因晶片實驗數據和動態模型識別方法，我們在三個胚胎幹細胞分化成的多巴胺神經元發展過程的假定的蛋白質網路下，可以修整候選的蛋白質網絡來取得精確的蛋白質網絡。藉由比較三個分化條件下的精確蛋白質網絡，代表胚胎幹細胞分化成多巴胺神經元的蛋白質網絡的轉變階段，我們得到更切實的動態蛋白質網絡伴隨者顯著的蛋白質網路的變化。然後，利用Reactome註解資料庫，富集的功能模塊將會在差異的蛋白質網絡被分別出來。從差異的蛋白質網路的角度，發現對多巴胺神經元發育的重要機制，我們專注在四個值得注意的胚胎幹細胞分化的生物模塊，如發育生物過程、免疫系統、凋亡和止血。從這些功能模塊和信路通路分析，我們推測顯著的多巴胺發展信號路徑，如血管新生、TGF-β信號通路、PI3激酶通路、Wnt信號通路，這些發現可以提供更多分化的分子機制的見解。我們建議的分化蛋白質網絡設計可以為藥物治療提供有用的策略，並促進對帕金森氏症新的藥物靶標發展。

Abstract
In this study, we focus on investigating significant functional modules and signal pathways in differential process from human embryonic stem cells (hESCs) to dopamine (DA) neurons. By data integration and mining, we can construct a candidate protein-protein interaction (PPI) network for expansion and differentiation process of hESCs. Then, based on microarray data in three differentiation conditions of hESCs and dynamic model identification method, we could prune the false positive PPIs in candidate PPI network to obtain refined PPI networks in three differentiation processes of hESCs to the development of DA neurons. By comparing three refined PPI networks at three differentiation conditions, which represent three transition stages of differential PPI network in differentiation of hESCs to DA neurons, we obtain more realistic dynamic PPI networks as PPI network reconfiguration with significant PPI changes between three stages of differentiation process. Then enriched functional modules among these significant changes in differential PPI networks were investigated by Reactome annotation. Four noteworthy biological functions in hESCs differentiation, such as developmental biology, immune system, apotosis and hemostasis were found on important developmental mechanism of DA neuron from the perspective of differential PPI network. From these functional modules, we speculate that significant DA developmental pathways, like Angiogenesis, TGF-beta signaling pathway, PI3 kinase pathway, Wnt signaling pathway, can provide more insights into the molecular mechanisms of differentiation. The proposed differential PPI network scheme can provide useful strategies for medical therapy and facilitate the development of new drug targets for Parkinson's disease.

Reference:

[1] W. Dauer and S. Przedborski, "Parkinson's disease: mechanisms and models," Neuron, vol. 39, pp. 889-909, 2003.
[2] C. R. Freed, et al., "Transplantation of embryonic dopamine neurons for severe Parkinson's disease," New England Journal of Medicine, vol. 344, pp. 710-719, 2001.
[3] J. H. Kim, et al., "Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease," Nature, vol. 418, pp. 50-56, 2002.
[4] M. Joksimovic, et al., "Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis," Nature neuroscience, vol. 12, pp. 125-131, 2009.
[5] M. Placzek and J. Briscoe, "The floor plate: multiple cells, multiple signals," Nature Reviews Neuroscience, vol. 6, pp. 230-240, 2005.
[6] R. Kittappa, et al., "The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age," PLoS biology, vol. 5, p. e325, 2007.
[7] Y. Ono, et al., "Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells," Development, vol. 134, pp. 3213-3225, 2007.
[8] S. Bonilla, et al., "Identification of midbrain floor plate radial glia‐like cells as dopaminergic progenitors," Glia, vol. 56, pp. 809-820, 2008.
[9] C. W. Pouton and J. M. Haynes, "Embryonic stem cells as a source of models for drug discovery," Nature Reviews Drug Discovery, vol. 6, pp. 605-616, 2007.
[10] S. Kriks, et al., "Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson/'s disease," Nature, 2011.
[11] N. Lyashenko, et al., "Differential requirement for the dual functions of [beta]-catenin in embryonic stem cell self-renewal and germ layer formation," Nature cell biology, vol. 13, pp. 753-761, 2011.
[12] J. A. Thomson, et al., "Embryonic stem cell lines derived from human blastocysts," science, vol. 282, pp. 1145-1147, 1998.
[13] B. D. MacArthur, et al., "Systems biology of stem cell fate and cellular reprogramming," Nature Reviews Molecular Cell Biology, vol. 10, pp. 672-681, 2009.
[14] Y. Sakamoto and G. Kitagawa, Akaike information criterion statistics: Kluwer Academic Publishers, 1987.
[15] C. Stark, et al., "BioGRID: a general repository for interaction datasets," Nucleic acids research, vol. 34, pp. D535-D539, 2006.
[16] M. Ashburner, et al., "Gene Ontology: tool for the unification of biology," Nature genetics, vol. 25, p. 25, 2000.
[17] D. Croft, et al., "Reactome: a database of reactions, pathways and biological processes," Nucleic acids research, vol. 39, pp. D691-D697, 2011.
[18] B. M. Bolstad, et al., "A comparison of normalization methods for high density oligonucleotide array data based on variance and bias," Bioinformatics, vol. 19, pp. 185-193, 2003.
[19] D. Mutch, et al., "The limit fold change model: a practical approach for selecting differentially expressed genes from microarray data," BMC bioinformatics, vol. 3, p. 17, 2002.
[20] Y. C. Wang and B. S. Chen, "Integrated cellular network of transcription regulations and protein-protein interactions," BMC systems biology, vol. 4, p. 20, 2010.
[21] J.-S. R. Jang, et al., Neuro-fuzzy and soft computing : a computational approach to learning and machine intelligence. Upper Saddle River, NJ: Prentice Hall, 1997.
[22] Z. Bar-Joseph, et al., "A new approach to analyzing gene expression time series data," presented at the Proceedings of the sixth annual international conference on Computational biology, Washington, DC, USA, 2002.
[23] C. De Boor, A practical guide to splines : with 32 figures, Rev. ed. New York: Springer, 2001.
[24] T. F. Orntoft, et al., "Genome-wide study of gene copy numbers, transcripts, and protein levels in pairs of non-invasive and invasive human transitional cell carcinomas," Molecular & Cellular Proteomics, vol. 1, pp. 37-45, Jan 2002.
[25] J. R. S. Newman, et al., "Single-cell proteomic analysis of S-cerevisiae reveals the architecture of biological noise," Nature, vol. 441, pp. 840-846, Jun 15 2006.
[26] R. Johansson, System modeling and identification. Englewood Cliffs, NJ: Prentice Hall, 1993.
[27] H. Akaike, "New Look at Statistical-Model Identification," Ieee Transactions on Automatic Control, vol. Ac19, pp. 716-723, 1974.
[28] H. Akaike, "Factor analysis and AIC," Psychometrika, vol. 52, pp. 317-332, 1987.
[29] D. R. Hyduke and B. O. Palsson, "Towards genome-scale signalling-network reconstructions," Nat Rev Genet, vol. 11, pp. 297-307, Feb 23 2010.
[30] G. Joshi-Tope, et al., "Reactome: a knowledgebase of biological pathways," Nucleic acids research, vol. 33, pp. D428-D432, 2005.
[31] P. D. Thomas, et al., "PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification," Nucleic acids research, vol. 31, pp. 334-341, 2003.
[32] A. L. Barabasi and Z. N. Oltvai, "Network biology: understanding the cell's functional organization," Nat Rev Genet, vol. 5, pp. 101-13, Feb 2004.
[33] R. H. Yuan, et al., "Overexpression of KIAA0101 predicts high stage, early tumor recurrence, and poor prognosis of hepatocellular carcinoma," Clinical Cancer Research, vol. 13, pp. 5368-5376, 2007.
[34] F. Simpson, et al., "The PCNA-associated factor KIAA0101/p15PAF binds the potential tumor suppressor product p33ING1b," Experimental cell research, vol. 312, pp. 73-85, 2006.
[35] J. Lu, et al., "Defined culture conditions of human embryonic stem cells," Proceedings of the National Academy of Sciences, vol. 103, pp. 5688-5693, 2006.
[36] R. H. Xu, et al., "Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells," Nature Methods, vol. 2, pp. 185-190, 2005.
[37] Q. Zhao, et al., "Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis," Developmental dynamics, vol. 209, pp. 377-386, 1997.
[38] B. Bakkaloglu, et al., "Molecular Cytogenetic Analysis and Resequencing of< i> Contactin Associated Protein-Like 2</i> in Autism Spectrum Disorders," The American Journal of Human Genetics, vol. 82, pp. 165-173, 2008.
[39] A. Gdalyahu, et al., "DCX, a new mediator of the JNK pathway," The EMBO journal, vol. 23, pp. 823-832, 2004.
[40] X. H. Jaglin, et al., "Mutations in the β-tubulin gene TUBB2B result in asymmetrical polymicrogyria," Nature genetics, vol. 41, pp. 746-752, 2009.
[41] S. J. Holland, et al., "Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells," The EMBO journal, vol. 16, pp. 3877-3888, 1997.
[42] M. R. Bekheirnia, et al., "Genotype–Phenotype Correlation in X-Linked Alport Syndrome," Journal of the American Society of Nephrology, vol. 21, pp. 876-883, 2010.
[43] V. Ramesh, "Merlin and the ERM proteins in Schwann cells, neurons and growth cones," Nature Reviews Neuroscience, vol. 5, pp. 462-470, 2004.
[44] L. Li, et al., "Human Embryonic Stem Cells Possess Immune‐Privileged Properties," Stem Cells, vol. 22, pp. 448-456, 2004.
[45] M. Drukker and N. Benvenisty, "The immunogenicity of human embryonic stem-derived cells," Trends in biotechnology, vol. 22, pp. 136-141, 2004.
[46] R. Medzhitov, "Recognition of microorganisms and activation of the immune response," Nature, vol. 449, pp. 819-26, Oct 18 2007.
[47] C. Carter, "Alzheimer's disease plaques and tangles: Cemeteries of a Pyrrhic victory of the immune defence network against herpes simplex infection at the expense of complement and inflammation-mediated neuronal destruction," Neurochemistry international, vol. 58, pp. 301-320, 2011.
[48] T. Minamino, et al., "MEKK1 suppresses oxidative stress-induced apoptosis of embryonic stem cell-derived cardiac myocytes," Proceedings of the National Academy of Sciences, vol. 96, p. 15127, 1999.
[49] A. D. Pyle, et al., "Neurotrophins mediate human embryonic stem cell survival," Nature biotechnology, vol. 24, pp. 344-350, 2006.
[50] M. Pesce, et al., "Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis)," Development, vol. 118, pp. 1089-1094, 1993.
[51] H. P. Gerber, et al., "VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism," Nature, vol. 417, pp. 954-958, 2002.
[52] M. Dean, et al., "Tumour stem cells and drug resistance," Nature Reviews Cancer, vol. 5, pp. 275-284, 2005.
[53] S. K. Ismail and J. R. Higgins, "Hemostasis in pre-eclampsia," Semin Thromb Hemost, vol. 37, pp. 111-7, 2011.
[54] C. J. Lockwood, et al., "Decidual Cell‐Expressed Tissue Factor Maintains Hemostasis in Human Endometrium," Annals of the New York Academy of Sciences, vol. 943, pp. 77-88, 2001.
[55] A. Luttun and P. Verhamme, "Keeping your vascular integrity: What can we learn from fish?," BioEssays, vol. 30, pp. 418-422, 2008.
[56] F. Fändrich, et al., "Embryonic stem cells share immune-privileged features relevant for tolerance induction," Journal of molecular medicine, vol. 80, pp. 343-350, 2002.
[57] C. Stephan and T. Brock, "Vascular endothelial growth factor, a multifunctional polypeptide," Puerto Rico health sciences journal, vol. 15, p. 169, 1996.
[58] M. A. Laflamme, et al., "Formation of human myocardium in the rat heart from human embryonic stem cells," The American journal of pathology, vol. 167, pp. 663-671, 2005.
[59] S. Saha, et al., "TGF [beta]/Activin/Nodal Pathway in Inhibition of Human Embryonic Stem Cell Differentiation by Mechanical Strain," Biophysical journal, vol. 94, pp. 4123-4133, 2008.
[60] K. Okkenhaug and B. Vanhaesebroeck, "PI3K in lymphocyte development, differentiation and activation," Nature Reviews Immunology, vol. 3, pp. 317-330, 2003.
[61] Y. Muroyama, et al., "Wnt signaling plays an essential role in neuronal specification of the dorsal spinal cord," Genes & development, vol. 16, pp. 548-553, 2002.
[62] G. Castelo-Branco, et al., "Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a," Proceedings of the National Academy of Sciences, vol. 100, p. 12747, 2003.
[63] X. Zeng, et al., "Dopaminergic differentiation of human embryonic stem cells," Stem Cells, vol. 22, pp. 925-940, 2004.
[64] T. C. Schulz, et al., "Differentiation of human embryonic stem cells to dopaminergic neurons in serum‐free suspension culture," Stem Cells, vol. 22, pp. 1218-1238, 2004.
[65] J. E. Lee, et al., "Canonical Wnt signaling through Lef1 is required for hypothalamic neurogenesis," Development, vol. 133, pp. 4451-4461, 2006.
[66] Y. Konishi, et al., "Cdh1-APC controls axonal growth and patterning in the mammalian brain," science, vol. 303, pp. 1026-1030, 2004.
[67] F. L'Episcopo, et al., "A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection," Molecular neurodegeneration, vol. 6, p. 49, 2011.
[68] S. Khosla, et al., "Building bone to reverse osteoporosis and repair fractures," The Journal of clinical investigation, vol. 118, p. 421, 2008.
[69] T. Shiraishi, et al., "Large-scale analysis of network bistability for human cancers," PLoS Comput Biol, vol. 6, p. e1000851, 2010.
[70] S. M. Friedman, et al., "Fidelity in protein synthesis. The role of the ribosome," J Biol Chem, vol. 243, pp. 5044-8, Oct 10 1968.
[71] A. Fathi, et al., "Comprehensive Gene Expression Analysis of Human Embryonic Stem Cells during Differentiation into Neural Cells," PloS one, vol. 6, p. e22856, 2011.
[72] J. A. King and W. M. Miller, "Bioreactor development for stem cell expansion and controlled differentiation," Current opinion in chemical biology, vol. 11, pp. 394-398, 2007.
[73] J. S. Meyer, et al., "Modeling early retinal development with human embryonic and induced pluripotent stem cells," Proceedings of the National Academy of Sciences, vol. 106, pp. 16698-16703, 2009.