单层贴壁法诱导人胚胎干细胞向平滑肌细胞分化及其机制初探

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英文题名：Differentiation of Human Embryonic Stem Cells into Smooth Muscle Cells in Adherent Monoculture and Preliminary Studies of the Underlying Mechanisms 作者：黄华荣 论文级别：博士 学科专业名称：生物物理学 中文关键词：胚胎干细胞 ; 平滑肌细胞 ; 单层贴壁 ; 体外分化 ; 全反式维甲酸 英文关键词：Embryonic stem cells ; Smooth musclce cells ; Adherent Monoculture ; Differentiaon in vitro ; atRA 学位年度：2006 导师：张铭 学科代码：071011 学位授予单位：浙江大学 论文提交日期：2006-10-01

摘要

胚胎干细胞(Embryonic stem cells，ESCs)是从囊胚中内细胞团(Inner mass cells，ICM)或原始生殖细胞(Primordial germ cells，PGCs)分离和克隆的多能性细胞，具有强大的自我更新能力和多向分化潜能，能够在体内或体外分化为成体中任何细胞类型。ES细胞作为体外理想的细胞模型，可以研究真核细胞在早期胚胎发育时期基因的表达和调控；也为未来的细胞治疗和再生医学带来了希望。
     平滑肌细胞作为构成血管壁的组织单元及维持血管张力的主要细胞成份，其分化和增殖在血管发生以及动脉粥样硬化、癌症和高血压等疾病中起着重要作用。培养的平滑肌很难稳定维持在一种表型，这在很大程度上限制了人们对其调控机理和途径的研究，利用可诱导的SMC体外分化系统对于进行SMC分子机制研究具有重要的价值。迄今为止，还没有体外定向诱导人胚胎干细胞分化为平滑肌的报道。此外人胚胎干细胞分化为其他细胞谱系也由于分化效率和均一性等问题也限制了其在医学治疗上的应用。
     本文我们首次将人胚胎干细胞(hESC)定向分化成平滑肌细胞(SMC)。通过单层贴壁途径，利用高浓度的维甲酸(10μMRA)诱导10天后，经流式细胞仪检测SMα-actin阳性细胞高达93％，结合细胞形态、RT-PCR、免疫荧光细胞化学、western blot、对促收缩剂刺激的响应、出现自发收缩等指标检测，显示诱导的细胞具有平滑肌细胞的典型特征。该方法的优点是不通过拟胚体途径、没有基质细胞共培养、不经过细胞分选过程，其诱导程序简单，诱导效率高。这种单因子，单层、高效率的体外诱导系统为研究平滑肌细胞发育的分子调控机理提供了一个极佳的平台，此外高纯度的诱导为将来细胞治疗相关疾病提供了良好的细胞资源。
     为了进一步探索RA在高效诱导hES分化为SMC的作用机制，我们从RA代谢系统的关键酶CYP26s入手，分析了诱导过程中CYP26s，RARs和RXRs等基因的表达变化，并研究维甲酸代谢抑制剂、RA加入的时间及其持续处理时间对SMC分化效率的影响。我们揭示了高浓度RA在hES细胞单层贴壁高效分化为SMC中的诱导和促凋亡的双重效应。在RA的作用下，由于RA的多生物学效应，ES细胞启动了向多种细胞谱系的分化(包括SMC谱系)，然而分化早期诱导出的各种祖细胞由于CYP26s的表达不同，失活RA的能力出现了明显差异，这些差异促使部分细胞(SMC前体细胞)由于CYP26s的高表达从而能够逃脱凋亡命运，最终使得ES细胞高效分化成为SMC。
     目前ES分化策略大部分是采用拟胚体的方法，拟胚体能够模拟胚胎发育早期的各种事件，但也存在诸如分析分选困难、细胞间有着复杂的相互作用等弊端。为使得分化为特定细胞的效率更高，并能方便研究驱动体外定向分化早期事情的分子机制，我们发展并完善了单层贴壁方法。单层贴壁进行分化研究的前提就是要获得高活力、高纯度的ES细胞，因此本实验利用胚胎干细胞与饲养层细胞贴壁速度的差异，建立了一种简单快速的分离MEF和ES细胞的差速贴壁方法。结果表明，在0.5％明胶包被的培养器皿中，差速贴壁1.5小时是分离ES细胞与MEF的最佳时间，此时流式细胞统计MEF仅占细胞群体2.4％。通过对差速贴壁前后的ES细胞进行碱性磷酸酶、单克隆形成率、ES细胞特异性标志、三胚层分化潜能等指标比较，证明纯化后的ES细胞仍保持高活力、未分化状态，这为后续进行ES细胞单层贴壁分化提供了条件。此方法不仅对ES细胞的干性无明显影响，可进行后续的分化实验，而且与其他去除饲养层的方法相比还具有高效、经济、快速、简单等优点。为通过单层贴壁途径进行ES细胞定向分化研究解决了细胞材料问题。
     在建立了单层贴壁分化ES细胞系统基础上，我们尝试将组蛋白脱乙酰酶抑制剂(丁酸钠)应用于神经细胞分化的研究，使得ES细胞定向分化为神经细胞的周期更短，效率更高，操作更简便。利用组蛋白脱乙酰酶抑制剂—NaB打破组蛋白乙酰化的平衡，促使染色质松散，从而抑制胚胎干细胞增殖，使起尽快步入分化状态，随即采用单层贴壁培养分化的方法结合添加神经诱导培养液N2827使其分化成神经细胞。结果表明此方法可获得高比例的神经细胞，在分化第4～6天时以nestin阳性的神经祖细胞为主，分化两周后就可产生大量表达GFAP的星性胶质细胞及表达β-tubulinⅢ的神经元。通过LAPS芯片检测诱导出的神经元具有放出惊厥样电位的功能。
Embryonic stem cells are pluripotent cells derived from the inner cell mass of pre-implantation mouse embryos. They have extensive selfrenewal capacity and are competent to differentiate into any cell type of the body in vivo and in vitro. It is a tractable cellular system to investigate cellular and genetic programming of early development. In terms of clinical benefit, stem cells are generating many hopes for future regenerative medicine.
     Smooth muscle cells (SMC) of the vascular system form an intriguing population of cells that are relevant for maintaining vascular tone and function. Proper regulation of vascular smooth muscle cell (SMC) differentiation and growth is critical for vasculogenesis and the maintenance of homeostasis in the mature vessel wall. Abnormal growth and proliferation of vascular smooth muscle cells (SMCs) is a key feature of vascular diseases such as atherosclerosis, restenosis, and hypertension. An in vitro system that recapitulates human SMC differentiation would be invaluable for exploring the environmental cues and molecular mechanisms leading to the human diseases. Various types of cell lineages but not involving SMC have been derived from human embryonic stem cells through directed in vitro differentiation, however, the differentiation efficiency and the homogeneity of the differentiated cells remained a major issue for potential application of these cells in therapeutic medicine.
     In this study, we have established an innovative culture system for the efficient differentiation of smooth muscle cells from human embryonic stem (ES) cells by monolayer adherent culture without feeder cells, embryoid bodies or cell-sorting processes. The hESCs were cultivated in differentiation medium containing 10μM of RA, more than 93 percent of all the cells expressed SMα-actin and 64 percent of all the cells expressed SM-MHC after 15 days. Combining to detect some parameters, such as cell appearance, RT-PCR, immunofluorescence, western blot, responding to agonist, spontaneous contraction, it shows hESCs-derived cells has the typical characteristic of the smooth muscle cell. The advantage of this system includes simple procedure and high efficiency. This inducible system (single factor, the monolayer, high efficiency) in vitro provide a very good platform for studying the molecular mechanism controlling SMC differentiation. Furthermore, high pure hESCs-derived SMC would provide a good cell resource for cell therapy in the future.
     Through the RT-PCR analysis for expression of cyp26s, RARs and RXRs in induced process, study retinoic acid metabolism blocking agent's influence to this monolayer system and time-dependent effects of RA on the SMC Differentiation of hESCs in vitro, we found that the high efficiency of the SMC differentiation in this system appears to result from double effects of differentiation-inducing and apoptotic of high atRA on the starting hESCs. Because of RA's multi-biological effects, the ES cells started differentiation toward various cell lineages (include SMC lineage) under RA. however progenitor cells in early differentiation appeared obvious difference about the ability with RA inactivation due to their expression dissimilarity of the CYP26s. These differences urged parts of cells (SMC progenitor cells) can succeed in escaping the destiny of apoptosis because of the high expression of the CYP26s. In the and, it make the hES cells differentiate SMC efficiently.
     To drive differentiation of mouse embryonic stem (ES) cells, various culture protocols have been previously developed that most require the formation of embryoid bodies or co-culture, Considering that, in terms of cell to-cell interactions, one-dimensional culture introduces lower complexity to the system compared to three-dimensional EBs, we reasoned that differentiation protocols involving only monolayer culture must be developed in order to: (1) make large scale production of neurodifferentiated cells more effective and to (2) allow for analyzing the molecular mechanisms that drive early events of neural differentiation in vitro.
     A significant problem for monolayer adherent differentiation is how to gain pure ES cells. We established a method of separating murine embryonic fibroblast from embryonic stem cells. By comparing the different adhesion rate of MEF and ES cells to find out the opitimum time for MEF removing. The result shows that 1.5h and 0.5% gelatin concentration is the opitimum condition for MEF removing. Furthermore, the ES cells which have been purified have the same plating efficiency and the ablity of three germ layers differentiation as the unpurified ES cells. After differential adhesion there is strong ALP activity in ES cells. All the results show that the purified ES cells are still in the state of undifferentiation and maintain the pluripotent.
     On the basis of establishing the ESC differentiation system by monolayer adherent culture, we try to apply sodium butyrate to research neural differentiation. It make the period of the ESC direct differentiate into neuron shorter, the efficiency is higher, manipulation is more simple. Use HDAC inhibitors (NaB) to breaks the balance of histone acetylation, make chromatin loose, thus repress embryo stem cells to propagate, make it turn into differentiation appearance as soon as possible, induce medium N2B27 was adopts by monolayer adherent culture immediately. The results show that high proportion of neuron were obtained by this method, there are mostly nestin-positive neural progenitor cell at 4-6 days, and ESCs were differentiated into GFAP-positive gails and neuron that expresβ-tubulin III after two weeks. ESCs-derived neuron have the function that let out electric potential by LAPS chip examination.

引文

1 Pardanaud L, Altmann C, Kitos P, Dieterlen-Lievre F, Buck CA. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development 1987; 100:339-349.
    2 DeRuiter MC, Poelmann RE, VanMunsteren JC, Mironov V, Markwald RR, Gittenberger-de Groot AC. Embryonic endothelial cells transdifferentiate into mesenchymal cells expressing smooth muscle actins in vivo and in vitro. Circ Res 1997;80:444-451.
    3 Risau W. Mechanisms of angiogenesis. Nature 1997;386:671-674.
    4 Vrancken Peeters MP, Gittenberger-de Groot AC, Mentink MM, Poelmann RE. Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium. Anat Embryol (Berl) 1999;199:367-378.
    5 Dettman RW, Denetclaw W, Jr., Ordahl CP, Bristow J. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol 1998;193:169-181.
    6 Rosenquist TH, Beall AC. Elastogenic cells in the developing cardiovascular system. Smooth muscle, nonmuscle, and cardiac neural crest. Ann N Y Acad Sci 1990;588:106-119.
    7 Le Lievre CS, Le Douarin NM. Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos. J Embryol Exp Morphol 1975; 34: 125-154.
    8 Bergwerff M, Verbeme ME, DeRuiter MC, Poelmann RE, Gittenberger-de Groot AC. Neural crest cell contribution to the developing circulatory system: implications for vascular morphology? Circ Res 1998;82:221-231.
    9 Miyagawa-Tomita S, Waldo K, Tomita H, Kirby ML. Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras. Am J Anat 1991;192:79-88.
    10 Waldo KL, Kirby ML. Cardiac neural crest contribution to the pulmonary artery and sixth aortic arch artery complex in chick embryos aged 6 to 18 days. Anat Rec 1993;237:385-399.
    11 Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 2002;8:403-409.
    12 Shimizu K, Sugiyama S, Aikawa M, Fukumoto Y, Rabkin E, Libby P, Mitchell RN. Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001;7:738-741.
    13 Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996; 183:1797-1806.
    14 Wakino S, Kintscher U, Kim S, Jackson S, Yin F, Nagpal S, Chandraratna RA, Hsueh WA, Law RE. Retinoids inhibit proliferation of human coronary smooth muscle cells by modulating cell cycle regulators. Arterioscler Thromb Vase Biol 2001; 21: 746-751.
    15 Majka SM, Jackson KA, Kienstra KA, Majesky MW, Goodell MA, Hirschi KK. Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration. J Clin Invest 2003; 111:71-79.
    16 Hillebrands JL, Klatter FA, van den Hurk BM, Popa ER, Nieuwenhuis P, Rozing J. Origin of neointimal endothelium and alpha-actin-positive smooth muscle cells in transplant arteriosclerosis. J Clin Invest 2001;107:1411-1422.
    17 Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation 2002; 106:1199-1204.
    18 Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, Dietrich H, Xu Q. Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation 2002; 106:1834-1839.
    19 Li J, Han X, Jiang J, Zhong R, Williams GM, Pickering JG, Chow LH. Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice. Am J Pathol 2001; 158:1943-1947.
    20 Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123-134.
    21 Paranya G, Vineberg S, Dvorin E, Kaushal S, Roth SJ, Rabkin E, Schoen FJ, Bischoff J. Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro. Am J Pathol 2001;159:1335-1343.
    22 Frid MG, Kale VA, Stenmark KR. Mature vascular endothelium can give rise to smooth muscle cells via endothelial-mesenchymal transdifferentiation: in vitro analysis. Circ Res 2002;90:1189-1196.
    23 Ishisaki A, Hayashi H, Li AJ, lmamura T. Human umbilical vein endothelium-derived cells retain potential to differentiate into smooth muscle-like cells. J Biol Chem 2003;278:1303-1309.
    24 Bochaton-Piallat ML, Ropraz P, Gabbiani F, Gabbiani G. Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol 1996;16:815-820.
    25 Ehler E, Jat PS, Noble MD, Citi S, Draeger A. Vascular smooth muscle cells of H-2Kb-tsA58 transgenic mice. Characterization of cell lines with distinct properties. Circulation 1995;92:3289-3296.
    26 Chamley-Campbell JH, Campbell GR. What controls smooth muscle phenotype? Atherosclerosis 1981;40:347-357.
    27 Bergwerff M, DeRuiter MC, Poelmann RE, Gittenberger-de Groot AC. Onset of elastogenesis and downregulation of smooth muscle actin as distinguishing phenomena in artery differentiation in the chick embryo. Anat Embryol (Berl) 1996; 194:545-557.
    28 Bochaton-Piallat ML, Gabbiani F, Redard M, Desmouliere A, Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathoi 1995; 146:1059-1064.
    29 Slomp J, Gittenberger-de Groot AC, Glukhova MA, Conny van Munsteren J, Kockx MM, Schwartz SM, Koteliansky VE. Differentiation, dedifferentiation, and apoptosis of smooth muscle cells during the development of the human ductus arteriosus. Arterioscler Thromb Vasc Biol 1997; 17: 1003-1009.
    30 Kockx MM, Cambier BA, Bonier HE, De Meyer GR, Declercq SC, van Cauwelaert PA, Bultinck J. Foam cell replication and smooth muscle cell apoptosis in human saphenous vein grafts. Histopathology 1994;25:365-371.
    31 Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest 1995;95:2266-2274.
    32 Chamley-Campbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev 1979;59:1-61.
    33 Hayashi K, Saga H, Chimori Y, Kimura K, Yamanaka Y, Sobue K. Differentiated phenotype of smooth muscle cells depends on signaling pathways through insulin-like growth factors and phosphatidylinositol 3-kinase. J Biol Chem 1998;273:28860-28867.
    34 Lau CL, Chacko S. Identification of two types of smooth muscle cells from rabbit urinary bladder. Tissue Cell 1996;28:339-355.
    35 Shanahan CM, Weissberg PL. Smooth muscle cell heterogeneity: patterns of gene expression in vascular smooth muscle cells in vitro and in vivo. Arterioscler Thromb Vase Biol 1998;18:333-338.
    36 Shanahan CM, Weissberg PL, Metcalfe JC. Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ Res 1993;73:193-204.
    37 Halayko AJ, Camoretti-Mercado B, Forsythe SM, Vieira JE, Mitchell RW, Wylam ME, Hershenson MB, Solway J. Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am J Physiol 1999;276:L197-206.
    38 Halayko AJ, Rector E, Stephens NL. Characterization of molecular determinants of smooth muscle cell heterogeneity. Can J Physiol Pharmacol 1997;75:917-929.
    39 Halayko AJ, Salari H, Ma X, Stephens NL. Markers of airway smooth muscle cell phenotype. Am J Physiol 1996;270:L1040-1051.
    40 Hayashi K, Takahashi M, Kimura K, Nishida W, Saga H, Sobue K. Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells. J Cell Biol 1999; 145:727-740.
    41 Archer SL, Huang JM, Reeve HL, Hampi V, Tolarova S, Michelakis E, Weir EK. Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ Res 1996;78:431-442.
    42 Frid MG, AIdashev AA, Dempsey EC, Stenmark KR. Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities. Circ Res 1997;81:940-952.
    43 Frid MG, Dempsey EC, Durmowicz AG, Stenmark KR. Smooth muscle cell heterogeneity in pulmonary and systemic vessels. Importance in vascular disease. Arterioscler Thromb Vasc Biol 1997;17:1203-1209.
    44 Letourneur D, Caleb BL, Castellot JJ, Jr. Heparin binding, internalization, and metabolism in vascular smooth muscle cells: Ⅱ. Degradation and secretion in sensitive and resistant cells. J Cell Physiol 1995; 165:687-695.
    45 Mitchell JJ, Reynolds SE, Leslie KO, Low RB, Woodcock-Mitchell J. Smooth muscle cell markers in developing rat lung. Am J Respir Cell Mol Biol 1990;3:515-523.
    46 Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 1995;75:487-517.
    47 Gimona M, Sparrow MP, Strasser P, Herzog M, Small JV. Calponin and SM 22 isoforms in avian and mammalian smooth muscle. Absence of phosphorylation in vivo. Eur J Biochem 1992;205:1067-1075.
    48 Topouzis S, Majesky MW. Smooth muscle lineage diversity in the chick embryo. Two types of aortic smooth muscle cell differ in growth and receptor-mediated transcriptional responses to transforming growth factor-beta. Dev Biol 1996; 178:430-445.
    49 Halayko AJ, Rector E, Stephens NL. Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition. Am J Physiol 1998;274:L17-25.
    50 Bacakova L, Pellicciari C, Bottone MG, Lisa V, Mares V. A sex-related difference in the hypertrophic versus hyperplastic response of vascular smooth muscle cells to repeated passaging in culture. Histol Histopathol 2001;16:675-684.
    51 Daemen MJ, De Mey JG. Regional heterogeneity of arterial structural changes. Hypertension 1995;25:464-473.
    52 Halayko AJ, Stephens NL. Potential role for phenotypic modulation of bronchial smooth muscle cells in chronic asthma. Can J Physiol Pharmacol 1994;72:1448-1457.
    53 Liddell RA, Syms M, McHugh KM. Heterogeneous isoactin gene expression in the adult rat gastrointestinal tract. Gastroenterology 1993;105:347-356.
    54 Ma X, Li W, Stephens NL. Detection of two clusters of mechanical properties of smooth muscle along the airway tree. J Appl Physiol 1996;80:857-861.
    55 Godfraind-De Becker A, Gillis JM. Polarized light microscopy of the smooth muscle anococcygeus of the rat. Adv Exp Med Biol 1988;226:149-154.
    56 Kolbeck RC, Speir WA, Jr. Regional contractile responses in pulmonary artery to alpha-and beta-adrenoceptor agonists. Can J Physiol Pharmacol 1987;65:1165-1170.
    57 Osborn M, Caselitz J, Weber K. Heterogeneity of intermediate filament expression in vascular smooth muscle: a gradient in desmin positive cells from the rat aortic arch to the level of the arteria iliaca communis. Differentiation 1981;20:196-202.
    58 Sigurdsson SB, Chitano P, Stephens NL. The Schultz-Dale response of sensitized canine bronchial smooth muscle. Respir Physiol 1995;102:79-87.
    59 Zanellato AM, Borrione AC, Giuriato L, Tonello M, Scannapieco G, Pauletto P, Sartore S. Myosin isoforms and cell heterogeneity in vascular smooth muscle. Ⅰ. Developing and adult bovine aorta. Dev Biol 1990;141:431-446.
    60 Letourneur D, Caleb BL, Castellot JJ, Jr. Heparin binding, internalization, and metabolism in vascular smooth muscle cells: Ⅰ. Upregulation of heparin binding correlates with antiproliferative activity. J Cell Physiol 1995;165:676-686.
    61 Weissberg PL, Grainger DJ, Shanahan CM, Metcalfe JC. Approaches to the development of selective inhibitors of vascular smooth muscle cell proliferation. Cardiovasc Res 1993;27:1191-1198.
    62 Weir EK, Reeve HL, Cornfield DN, Tristani-Firouzi M, Peterson DA, Archer SL. Diversity of response in vascular smooth muscle cells to changes in oxygen tension. Kidney Int 1997;51:462-466.
    63 Reeve HL, Weir EK, Archer SL, Cornfield DN. A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent. Am J Physiol 1998;275:L1019-1025.
    64 Majack RA, Grieshaber NA, Cook CL, Weiser MC, McFall RC, Gdeshaber SS, Reidy MA, Reilly CF. Smooth muscle cells isolated from the neointima after vascular injury exhibit altered responses to platelet-derived growth factor and other stimuli. J Cell Physiol 1996;167:106-112.
    65 Barzu T, Pascal M, Maman M, Roque C, Lafont F, Rousselet A. Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: comparison between heparin-sensitive and heparin-resistant cultures. J Cell Physiol 1996; 167:8-21.
    66 Murry CE, Gipaya CT, Bartosek T, Benditt EP, Schwartz SM. Monoclonality of smooth muscle cells in human atherosclerosis. Am J Pathol 1997;151:697-705.
    67 Wohrley JD, Frid MG, Moiseeva EP, Orton EC, Belknap JK, Stenmark KR. Hypoxia selectively induces proliferation in a specific subpopulation of smooth muscle cells in the bovine neonatal pulmonary arterial media. J Clin Invest 1995;96:273-281.
    68 Eddinger TJ, Korwek AA, Meer DP, Sherwood JJ. Expression of smooth muscle myosin light chain 17 and unloaded shortening in single smooth muscle cells. Am J Physiol Cell Physiol 2000;278:C1133-1142.
    69 Meer DP, Eddinger TJ. Expression of smooth muscle myosin heavy chains and unloaded shortening in single smooth muscle cells. Am J Physiol 1997;273:C1259-1266.
    70 Madden JA, Vadula MS, Kurup VP. Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells. Am J Physiol 1992;263:L384-393.
    71 Durmowicz AG, Frid MG, Wohrley JD, Stenmark KR. Expression and localization of tropoelastin mRNA in the developing bovine pulmonary artery is dependent on vascular cell phenotype. Am J Respir Cell Mol Biol 1996;14:569-576.
    72 Benzakour O, Kanthou C, Kanse SM, Scully MF, Kakkar VV, Cooper DN. Evidence for cultured human vascular smooth muscle cell heterogeneity: isolation of clonal cells and study of their growth characteristics. Thromb Haemost 1996;75:854-858.
    73 Holifield B, Helgason T, Jemelka S, Taylor A, Navran S, Allen J, Seidel C. Differentiated vascular myocytes: are they involved in neointimal formation? J Clin Invest 1996;97:814-825.
    74 Mironov AA, Rekhter MD, Kolpakov VA, Andreeva ER, Polishchuk RS, Bannykh SI, Filippov SV, Peretjatko LP, Kulida LV, Orekhov AN. Heterogeneity of smooth muscle cells in embryonic human aorta. Tissue Cell 1995;27:31-38.
    75 Neylon CB, Avdonin PV, Dilley RJ, Larsen MA, Tkachuk VA, Bobik A. Different electrical responses to vasoactive agonists in morphologically distinct smooth muscle cell types. Circ Res 1994;75:733-741.
    76 Villaschi S, Nicosia RF, Smith MR. Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity. In Vitro Cell Dev Biol Anim 1994;30A:589-595.
    77 Neuville P, Geinoz A, Benzonana G, Redard M, Gabbiani F, Ropraz P, Gabbiani G. Cellular retinol-binding protein-1 is expressed by distinct subsets of rat arterial smooth muscle cells in vitro and in vivo. Am J Pathol 1997;150:509-521.
    78 Firulli AB, Miano JM, Bi W, Johnson AD, Casscells W, Olson EN, Schwarz JJ. Myocyte enhancer binding factor-2 expression and activity in vascular smooth muscle cells. Association with the activated phenotype. Circ Res 1996;78:196-204.
    79 Klein T, Ling Z, Heimberg H, Madsen OD, Heiler RS, Serup P. Nestin is expressed in vascular endothelial cells in the adult human pancreas. J Histochem Cytochem 2003;51:697-706.
    80 Kim S, Ip HS, Lu MM, Clendenin C, Parmacek MS. A serum response factor-dependent transcriptional regulatory program identifies distinct smooth muscle cell sublineages. Mol Cell Biol 1997; 17:2266-2278.
    81 Madsen CS, Regan CP, Hungerford JE, White SL, Manabe I, Owens GK. Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence. Circ Res 1998;82:908-917.
    82 Miano JM, Olson EN. Expression of the smooth muscle cell calponin gene marks the early cardiac and smooth muscle cell lineages during mouse embryogenesis. J Biol Chem 1996;271:7095-7103.
    83 Campbell GR, Campbell JH. Smooth muscle phenotypic changes in arterial wall homeostasis: implications for the pathogenesis of atherosclerosis. Exp Mol Pathol 1985;42:139-162.
    84 Majesky MW, Giachelli CM, Reidy MA, Schwartz SM. Rat carotid neointimai smooth muscle cells reexpress a developmentally regulated mRNA phenotype during repair of arterial injury. Circ Res 1992;71:759-768.
    85 Zaugg P, Djonov V, Fuchtbauer EM, Draeger A. Sorting of murine vascular smooth muscle cells during wound healing in the chicken chorioallantoic membrane. Exp Cell Res 1999;253:599-606.
    86 Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 1993; 148:720-726.
    87 Gizycki MJ, Adelroth E, Rogers AV, O'Byrne PM, Jeffery PK. Myofibroblast involvement in the allergen-induced late response in mild atopic asthma. Am J Respir Cell Mol Biol 1997; 16:664-673.
    88 Holgate ST, Davies DE, Lackie PM, Wilson SJ, Puddicombe SM, Lordan JL. Epithelial-mesenchymal interactions in the pathogenesis of asthma. J Allergy Clin Immunol 2000; 105:193-204.
    89 Gabbiani G, Schmid E, Winter S, Chaponnier C, de Ckhastonay C, Vandekerckhove J, Weber K, Franke WW. Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific alpha-type actin. Proc Natl Acad Sci U S A 1981;78:298-302.
    90 Hungerford JE, Owens GK, Argraves WS, Little CD. Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev Biol 1996; 178:375-392.
    91 Mack CP, Owens GK. Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions. Circ Res 1999;84:852-861.
    92 Arimura C, Suzuki T, Yanagisawa M, lmamura M, Hamada Y, Masaki T. Primary structure of chicken skeletal muscle and fibroblast aipha-actinins deduced from cDNA sequences. Eur J Biochem 1988; 177:649-655.
    93 Babij P, Kelly C, Periasamy M. Characterization of a mammalian smooth muscle myosin heavy-chain gene: complete nucleotide and protein coding sequence and analysis of the 5' end of the gene. Proc Natl Acad Sci U S A 1991;88:10676-10680.
    94 Lazard D, Sastre X, Frid MG, Glukhova MA, Thiery JP, Koteliansky VE. Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc Natl Acad Sci U S A 1993;90:999-1003.
    95 Miano JM, Cserjesi P, Ligon KL, Periasamy M, Olson EN. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res 1994;75:803-812.
    96 Duband JL, Gimona M, Scatena M, Sartore S, Small JV. Calponin and SM 22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development. Differentiation 1993;55:1-11.
    97 Miano JM, Carlson MJ, Spencer JA, Misra RP. Serum response factor-dependent regulation of the smooth muscle calponin gene. J Biol Chem 2000;275:9814-9822.
    98 Strasser P, Gimona M, Moessler H, Herzog M, Small JV. Mammalian calponin. Identification and expression of genetic variants. FEBS Lett 1993;330:13-18.
    99 Duggal N, Schmidt-Kastner R, Hakim AM. Nestin expression in reactive astrocytes following focal cerebral ischemia in rats. Brain Res 1997;768:1-9.
    100 Li L, Miano JM, Cserjesi P, Olson EN. SM22 alpha, a marker of adult smooth muscle, is expressed in multiple myogenic lineages during embryogenesis. Circ Res 1996;78:188-195.
    101 Layne MD, Yet SF, Maemura K, Hsieh CM, Liu X, Ith B, Lee ME, Perrella MA. Characterization of the mouse aortic carboxypeptidase-like protein promoter reveals activity in differentiated and dedifferentiated vascular smooth muscle cells. Circ Res 2002;90:728-736.
    102 Watanabe M, Layne MD, Hsieh CM, Maemura K, Gray S, Lee ME, Jain MK. Regulation of smooth muscle cell differentiation by AT-rich interaction domain transcription factors Mrf2alpha and Mrf2beta. Circ Res 2002;91:382-389.
    103 Bolmont C, Lilienbaum A, Paulin D, Grimaud JA. Expression of desmin gene in skeletal and smooth muscle by in situ hybridization using a human desmin gene probe. J Submicrosc Cytol Pathol 1990;22:117-122.
    104 Mericskay M, Parlakian A, Porteu A, Dandre F, Bonnet J, Paulin D, Li Z. An overlapping CArG/octamer element is required for regulation of desmin gene transcription in arterial smooth muscle cells. Dev Biol 2000;226:192-208.
    105 Frid MG, Shekhonin BV, Koteliansky VE, Glukhova MA. Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and caiponin. Dev Biol 1992;153:185-193.
    106 Sobue K, Sellers JR. Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. J Biol Chem 1991;266:12115-12118.
    107 Yano H, Hayashi K, Momiyama T, Saga H, Haruna M, Sobue K. Transcriptional regulation of the chicken caldesmon gene. Activation of gizzard-type caldesmon promoter requires a CArG box-like motif. J Biol Chem 1995;270:23661-23666.
    108 Gimona M, Furst DO, Small JV. Metavinculin and vinculin from mammalian smooth muscle: bulk isolation and characterization. J Muscle Res Cell Motil 1987;8:329-341.
    109 Herring BP, Lyons GE, Hoggatt AM, Gallagher PJ. Telokin expression is restricted to smooth muscle tissues during mouse development. Am J Physiol Cell Physiol 2001;280:C12-21.
    110 van der Loop FT, Schaart G, Timmer ED, Ramaekers FC, van Eys GJ. Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells. J Cell Biol 1996; 134:401-411.
    111 Hsieh CM, Yoshizumi M, Endege WO, Kho CJ, Jain MK, Kashiki S, de los Santos R, Lee WS, Perrella MA, Lee ME. APEG-1, a novel gene preferentially expressed in aortic smooth muscle cells, is down-regulated by vascular injury. J Biol Chem 1996;271:17354-17359.
    112 Jain MK, Fujita KP, Hsieh CM, Endege WO, Sibinga NE, Yet SF, Kashiki S, Lee WS, Perrella MA, Haber E, Lee ME. Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells. J Biol Chem 1996;271:10194-10199.
    113 Jain MK, Kashiki S, Hsieh CM, Layne MD, Yet SF, Sibinga NE, Chin MT, Feinberg MW, Woo I, Maas RL, Haber E, Lee ME. Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system. Circ Res 1998;83:980-985.
    114 Layne MD, Endege WO, Jain MK, Yet SF, Hsieh CM, Chin MT, Perrella MA, Blanar MA, Haber E, Lee ME. Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation. J Biol Chem 1998;273:15654-15660.
    115 Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res 1999;36:2-27.
    116 Sobue K, Hayashi K, Nishida W. Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation. Mol Cell Biochem 1999;190:105-118.
    117 Giachelli CM, Bae N, Almeida M, Denhardt DT, AIpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest 1993;92:1686-1696.
    118 Kuro-o M, Nagai R, Nakahara K, Katoh H, Tsai RC, Tsuchimochi H, Yazaki Y, Ohkubo A, Takaku F. cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis. J Biol Chem 1991;266:3768-3773.
    119 Bochaton-Piallat ML, CIowes AW, Clowes MM, Fischer JW, Redard M, Gabbiani F, Gabbiani G. Cultured arterial smooth muscle cells maintain distinct phenotypes when implanted into carotid artery. Arterioscler Thromb Vasc Biol 2001;21:949-954.
    120 Manfridi A, Brambilla D, Bianchi S, Mariotti M, Opp MR, Imeri L. Interleukin-lbeta enhances non-rapid eye movement sleep when microinjected into the dorsal raphe nucleus and inhibits serotonergic neurons in vitro. Eur J Neurosci 2003; 18:1041-1049.
    121 Aumailley M, Gayraud B. Structure and biological activity of the extracellular matrix. J Mol Meal 1998;76:253-265.
    122 Yamamoto M, Yamamoto K. [Regulation of differentiated properties of arterial smooth muscle cells]. Nippon Rinsho 1993;51: 1980-1986.
    123 Hedin U, Thyberg J. Plasma fibronectin promotes modulation of arterial smooth-muscle cells from contractile to synthetic phenotype. Differentiation 1987;33:239-246.
    124 Qin H, Ishiwata T, Wang R, Kudo M, Yokoyama M, Naito Z, Asano G. Effects of extracellular matrix on phenotype modulation and MAPK transduction of rat aortic smooth muscle cells in vitro. Exp Mol Pathol 2000;69:79-90.
    125 Roy J, Tran PK, Religa P, Kazi M, Henderson B, Lundmark K, Hedin U. Fibronectin promotes cell cycle entry in smooth muscle cells in primary culture. Exp Cell Res 2002; 273: 169-177.
    126 Roy J, Kazi M, Hedin U, Thyberg J. Phenotypic modulation of arterial smooth muscle cells is associated with prolonged activation of ERK 1/2. Differentiation 2001;67:50-58.
    127 Schauwienold D, Plum C, Helbing T, Voigt P, Bobbert T, Hoffmann D, Paul M, Reusch HP. ERK1/2-dependent contractile protein expression in vascular smooth muscle cells. Hypertension 2003;41:546-552.
    128 Moses S, Franzen A, Lovdahl C, Hultgardh-Nilsson A. Injury-induced osteopontin gene expression in rat arterial smooth muscle cells is dependent on mitogen-activated protein kinases ERK1/ERK2. Arch Biochem Biophys 2001;396:133-137.
    129 Taher TE, Derksen PW, de Boer OJ, Spaargaren M, Teeling P, van der Wal AC, Pals ST. Hepatocyte growth factor triggers signaling cascades mediating vascular smooth muscle cell migration. Biochem Biophys Res Commun 2002;298:80-86.
    130 Berrou E, Bryckaert M. Platelet-derived growth factor inhibits smooth muscle cell adhesion to fibronectin by ERK-dependent and ERK-independent pathways. J Biol Chem 2001;276:39303-39309.
    131 Stringa E, Knauper V, Murphy G, Gavrilovic J. Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells. J Cell Sci 2000;113(Pt 11):2055-2064.
    132 Kingsley K, Rust WL, Huff JL, Smith RC, Plopper GE. PDGF-BB enhances expression of, and reduces adhesion to, laminin-5 in vascular smooth muscle cells. Biochem Biophys Res Commun 2002; 294: 1017-1022.
    133 Stegemann JP, Nerem RM. Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture. Exp Cell Res 2003;283:146-155.
    134 Aubert J, Stavridis MP, Tweedie S, O'Reilly M, Vierlinger K, Li M, Ghazal P, Pratt T, Mason JO, Roy D, Smith A. Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Soxl-gfp knock-in mice. Proc Natl Acad Sci U S A 2003; 100 Suppl 1:11836-11841.
    135 Grassl ED, Oegema TR, Tranquillo RT. A fibrin-based arterial media equivalent. J Biomed Mater Res A 2003;66:550-561.
    136 Cheung P, Allis CD, Sassone-Corsi P. Signaling to chromatin through histone modifications. Cell 2000; 103:263-271.
    137 Marmorstein R, Roth SY. Histone acetyltransferases: function, structure, and catalysis. Curr Opin Genet Dev 2001; 11:155-161.
    138 Manabe I, Owens GK. CArG elements control smooth muscle subtype-specific expression of smooth muscle myosin in vivo. J Clin Invest 2001;107:823-834.
    139 Manabe I, Owens GK. Recruitment of serum response factor and hyperacetylation of histones at smooth muscle-specific regulatory regions during differentiation of a novel P19-derived in vitro smooth muscle differentiation system. Circ Res 2001;88:1127-1134.
    140 Ramirez S, Ait-Si-Ali S, Robin P, Trouche D, Harel-Bellan A. The CREB-binding protein (CBP) cooperates with the serum response factor for transactivation of the c-fos serum response element. J Biol Chem 1997;272:31016-31021.
    141 Qiu P, Li L. Histone acetylation and recruitment of serum responsive factor and CREB-binding protein onto SM22 promoter during SM22 gene expression. Circ Res 2002;90:858-865.
    142 Wada H, Hasegawa K, Morimoto T, Kakita T, Yanazume T, Sasayama S. A p300 protein as a coactivator of GATA-6 in the transcription of the smooth muscle-myosin heavy chain gene. J Biol Chem 2000;275:25330-25335.
    143 Aravind L, Koonin EV. SAP-a putative DNA-binding motif involved in chromosomal organization. Trends Biochem Sci 2000;25:112-114.
    144 Hirst SJ, Twort CH, Lee TH. Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype. Am J Respir Cell Mol Biol 2000;23:335-344.
    145 Chen S, Gardner DG. Retinoic acid uses divergent mechanisms to activate or suppress mitogenesis in rat aortic smooth muscle cells. J Clin Invest 1998;102:653-662.
    146 Peclo MM, Printseva O. Retinoic acid enhances the proliferation of smooth muscle cells. Experientia 1987;43:196-198.
    147 Anderson RJ, Ray CJ, Hattler BG. Retinoic acid regulation of renal tubular epithelial and vascular smooth muscle cell function. J Am Soc Nephrol 1998;9:773-781.
    148 Hayashi A, Suzuki T, Tajima S. Modulations of elastin expression and cell proliferation by retinoids in cultured vascular smooth muscle cells. J Biochem (Tokyo) 1995;117:132-136.
    149 Corbeil J, Rapaport E, Richman DD, Looney DJ. Antiproliferative effect of retinoid compounds on Kaposi's sarcoma cells. J Clin Invest 1994;93:1981-1986.
    150 Benson S, Padmanabhan S, Kurtz TW, Pershadsingh HA. Ligands for the peroxisome proliferator-activated receptor-gamma and the retinoid X receptor-alpha exert synergistic antiproliferative effects on human coronary artery smooth muscle cells. Mol Cell Biol Res Commun 2000;3:159-164.
    151 Hagiwara H, Inoue A, Nakajo S, Nakaya K, Kojima S, Hirose S. Inhibition of proliferation of chondrocytes by specific receptors in response to retinoids. Biochem Biophys Res Commun 1996;222:220-224.
    152 Christen T, Bochaton-Piallat ML, Neuville P, Rensen S, Redard M, van Eys G, Gabbiani G. Cultured porcine coronary artery smooth muscle cells. A new model with advanced differentiation. Circ Res 1999;85:99-107.
    153 Fukamauchi F, Mataga N, Wang YJ, Sato S, Yoshiki A, Kusakabe M. Tyrosine hydroxylase activity and its mRNA level in dopaminergic neurons of tenascin gene knockout mouse. Biochem Biophys Res Commun 1997;231:356-359.
    154 Kim HS, Aikawa M, Kimura K, Kuro-o M, Nakahara K, Suzuki T, Katoh H, Okamoto E, Yazaki Y, Nagai R. Ductus arteriosus. Advanced differentiation of smooth muscle cells demonstrated by myosin heavy chain isoform expression in rabbits. Circulation 1993;88:1804-1810.
    155 Miano JM, Topouzis S, Majesky MW, Olson EN. Retinoid receptor expression and all-trans retinoic acid-mediated growth inhibition in vascular smooth muscle cells. Circulation 1996;93:1886-1895.
    156 Pakala R, Benedict CR. RAR gamma agonists inhibit proliferation of vascular smooth muscle cells. J Cardiovasc Pharmacol 2000;35:302-308.
    157 Braunhut SJ, Palomares M. Modulation of endothelial cell shape and growth by retinoids. Microvasc Res 1991;41:47-62.
    158 Lansink M, van Bennekum AM, Blaner WS, Kooistra T. Differences in metabolism and isomerization of all-trans-retinoic acid and 9-cis-retinoic acid between human endothelial cells and hepatocytes. Eur J Biochem 1997;247:596-604.
    159 Thompson JN, Howell JM, Pitt GA, McLaughlin CI. The biological activity of retinoic acid in the domestic fowl and the effects of vitamin A deficiency on the chick embryo. Br J Nutr 1969;23:471-490.
    160 Heine UI, Roberts AB, Munoz EF, Roche NS, Sporn MB. Effects of retinoid deficiency on the development of the heart and vascular system of the quail embryo. Virchows Arch B Cell Pathoi Incl Mol Pathol 1985;50:135-152.
    161 Niederreither K, Subbarayan V, Dolle P, Chambon P. Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nat Genet 1999;21:444-448.
    162 Yasuda Y, Nishi N, Takahashi JA, Konishi H, Ohara I, Fujita H, Ohta M, Itoh N, Hatanaka M, Tanimura T. Induction of avascular yolk sac due to reduction of basic fibroblast growth factor by retinoic acid in mice. Dev Biol 1992;150:397-413.
    163 Lammer EJ, Chen DT, Hoar RM, Agnish ND, Benke PJ, Braun JT, Curry CJ, Fernhoff PM, Grix AW, Jr., Lott IT, et al. Retinoic acid embryopathy. N Engl J Med 1985;313:837-841.
    164 Sucov HM, Dyson E, Gumeringer CL, Price J, Chien KR, Evans RM. RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis. Genes Dev 1994;8:1007-1018.
    165 Kastner P, Grondona JM, Mark M, Gansmuller A, LeMeur M, Decimo D, Vonesch JL, Dolle P, Chambon P. Genetic analysis of RXR alpha developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis. Cell 1994;78:987-1003.
    166 Kastner P, Mark M, Chambon P. Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell 1995;83:859-869.
    167 Krezel W, Dupe V, Mark M, Dierich A, Kastner P, Chambon P. RXR gamma null mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/- mutant mice are viable. Proc Natl Acad Sci U S A 1996;93:9010-9014.
    168 Stenmark KR, Frid MG. Smooth muscle cell heterogeneity: role of specific smooth muscle cell subpopulations in pulmonary vascular disease. Chest 1998; 114:82S-90S.
    169 Li L, Miano JM, Mercer B, Olson EN. Expression of the SM22alpha promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells. J Cell Biol 1996; 132:849-859.
    170 Colbert MC, Kirby ML, Robbins J. Endogenous retinoic acid signaling colocalizes with advanced expression of the adult smooth muscle myosin heavy chain isoform during development of the ductus arteriosus. Circ Res 1996;78:790-798.
    171 Desai DS, Hirai S, Karnes WE, Jr., Niles RM, Ohno S. Cloning and characterization of the murine PKC alpha promoter: identification of a retinoic acid response element. Biochem Biophys Res Commun 1999;263:28-34.
    172 Blank RS, Swartz EA, Thompson MM, OIson EN, Owens GK. A retinoic acid-induced clonal cell line derived from multipotential P19 embryonal carcinoma cells expresses smooth muscle characteristics. Circ Res 1995;76:742-749.
    173 Suzuki T, Kim HS, Kurabayashi M, Hamada H, Fujii H, Aikawa M, Watanabe M, Watanabe N, Sakomura Y, Yazaki Y, Nagai R. Preferential differentiation of P19 mouse embryonal carcinoma cells into smooth muscle cells. Use of retinoic acid and antisense against the central nervous system-specific POU transcription factor Brn-2. Circ Res 1996;78:395-404.
    174 Yao CC, Breuss J, Pytela R, Kramer RH. Functional expression of the alpha 7 integrin receptor in differentiated smooth muscle cells. J Cell Sci 1997;110(Pt 13):1477-1487.
    175 Drab M, Hailer H, Bychkov R, Erdmann B, Lindschau C, Haase H, Morano I, Luft FC, Wobus AM. From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model. Faseb J 1997; 11:905-915.
    176 Hautmann MB, Thompson MM, Swartz EA, Olson EN, Owens GK. Angiotensin Ⅱ-induced stimulation of smooth muscle alpha-actin expression by serum response factor and the homeodomain transcription factor MHox. Circ Res 1997;81:600-610.
    177 Gollasch M, Haase H, Ried C, Lindschau C, Morano I, Luft FC, Hailer H. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. Faseb J 1998;12:593-601.
    178 Wright G, Wang S, Bailey G, Reichenbecher V, Wright GL. Effect of retinoic acid on contractile competence of vascular smooth muscle. Am J Physiol 1996;270:H1363-1370.
    179 Miano JM, Kelly LA, Artacho CA, Nuckolls TA, Piantedosi R, Blaner WS. all-Trans-retinoic acid reduces neointimal formation and promotes favorable geometric remodeling of the rat carotid artery after balloon withdrawal injury. Circulation 1998;98:1219-1227.
    180 Neuville P, Yan Z, Gidlof A, Pepper MS, Hansson GK, Gabbiani G, Sirsjo A. Retinoic acid regulates arterial smooth muscle cell proliferation and phenotypic features in vivo and in vitro through an RARalpha-dependent signaling pathway. Arterioscler Thromb Vasc Biol 1999;19:1430-1436.
    181 DeRose JJ, Jr., Madigan J, Umana JP, Prystowsky JH, Nowygrod R, Oz MC, Todd GJ. Retinoic acid suppresses intimai hyperplasia and prevents vessel remodeling following arterial injury. Cardiovasc Surg 1999;7:633-639.
    182 Chen J, He B, Zheng D, Zhang S, Liu J, Zhu S. All-trans retinoic acid reduces intimal thickening after balloon angioplasty in atherosclerotic rabbits. Chin Med J (Engl) 1999;112:121-123.
    183 Kumar A, Lindner V. Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. Arterioscler Thromb Vasc Biol 1997;17:2238-2244.
    184 James TW, Wagner R, White LA, Zwolak RM, Brinckerhoff CE. Induction of collagenase and stromelysin gene expression by mechanical injury in a vascular smooth muscle-derived cell line. J Cell Physiol 1993;157:426-437.
    185 Ross SA, McCaffery PJ, Drager UC, De Luca LM. Retinoids in embryonal development. Physiol Rev 2000;80:1021-1054.
    186 Johnson RJ, lida H, Alpers CE, Majesky MW, Schwartz SM, Pritzi P, Gordon K, Gown AM. Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation. J Clin Invest 1991;87:847-858.
    187 Miano JM, Vlasic N, Tota RR, Stemerman MB. Smooth muscle cell immediate-early gene and growth factor activation follows vascular injury. A putative in vivo mechanism for autocrine growth. Arterioscler Thromb 1993; 13:211-219.
    188 Fanjul A, Dawson MI, Hobbs PD, Jong L, Cameron JF, Harlev E, Graupner G, Lu XP, Pfahl M. A new class of retinoids with selective inhibition of AP-1 inhibits proliferation. Nature 1994;372:107-111.
    189 Takahashi N, Breitman TR. Retinoylation of proteins in leukemia, embryonal carcinoma, and normal kidney cell lines: differences associated with differential responses to retinoic acid. Arch Biochem Biophys 1991;285:105-110.
    190 Wiegman PJ, Barry WL, McPherson JA, McNamara CA, Gimple LW, Sanders JM, Bishop GG, Powers ER, Ragosta M, Owens GK, Sarembock IJ. All-trans-retinoic acid limits restenosis after balloon angioplasty in the focally atherosclerotic rabbit : a favorable effect on vessel remodeling. Arterioscler Thromb Vasc Biol 2000;20:89-95.
    1 Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154-156.
    2 Thomson JA, Marshall VS, Trojanowski JQ. Neural differentiation of rhesus embryonic stem cells. Apmis 1998;106:149-156; discussion 156-147.
    3 Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 1998;95:13726-13731.
    4 Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. Stem Cells 2001; 19:193-204.
    5 Bradley A, Evans M, Kaufman MH, Robertson E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 1984;309:255-256.
    6 Arenas E. Stem cells in the treatment of Parkinson's disease. Brain Res Bull 2002;57:795-808.
    7 Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985;87:27-45.
    8 Shen MM, Leder P. Leukemia inhibitory factor is expressed by the preimplantation uterus and selectively blocks primitive ectoderm formation in vitro. Proc Natl Acad Sci U S A 1992;89:8240-8244.
    9 Lake J, Rathjen J, Remiszewski J, Rathjen PD. Reversible programming of pluripotent cell differentiation. J Cell Sci 2000;113(Pt 3):555-566.
    10 Takahashi M, Hayashi K, Yoshida K, Ohkawa Y, Komurasaki T, Kitabatake A, Ogawa A, Nishida W, Yano M, Monden M, Sobue K. Epiregulin as a major autocrine/paracrine factor released from ERK- and p38MAPK-activated vascular smooth muscle cells. Circulation 2003;108:2524-2529.
    11 Ying QL, Stavridis M, Griffiths D, Li M, Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monocuiture. Nat Biotechnol 2003;21: 183-186.
    12 van Inzen WG, Peppelenbosch MP, van den Brand MW, Tertoolen LG, de Laat SW. Neuronal differentiation of embryonic stem cells. Biochim Biophys Acta 1996;1312:21-26.
    13 Pachernik J, Esner M, Bryja V, Dvorak P, Hampl A. Neural differentiation of mouse embryonic stem cells grown in monolayer. Reprod Nutr Dev 2002;42:317-326.
    14 Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 1998;125:1747-1757.
    15 Li X J, Du ZW, Zarnowska ED, Pankratz M, Hansen LO, Pearce RA, Zhang SC. Specification of motoneurons from human embryonic stem cells. Nat Biotechnol 2005;23:215-221.
    16 Sone M, Itoh H, Yamashita J, Yurugi-Kobayashi T, Suzuki Y, Kondo Y, Nonoguchi A, Sawada N, Yamahara K, Miyashita K, Park K, Shibuya M, Nito S, Nishikawa S, Nakao K. Different differentiation kinetics of vascular progenitor cells in primate and mouse embryonic stem cells. Circulation 2003;107:2085-2088.
    17 Xu H, Fan X, Tang J, Zhou G, Yang L, Wu X, Liu S, Qu J, Yang H. A modified method for generation of neural precursor cells from cultured mouse embryonic stem cells. Brain Res Brain Res Protoc 2005;15:52-58.
    18 Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2000;97:11307-11312.
    19 王建，赵惠萍，谢常青，林戈，杨胜，聂东宋，王绮如，卢光绣．人类胚胎干细胞向造血细胞和内皮细胞诱导分化的初步研究．中国实验血液学杂志 2005；13：222-228．
    20 Yokote K, Take A, Nakaseko C, Kobayashi K, Fujimoto M, Kawamura H, Maezawa Y, Nishimura M, Mori S, Saito Y. Bone marrow-derived vascular cells in response to injury. J Atheroscler Thromb 2003;10:205-210.
    21 Miles GB, Yohn DC, Wichterle H, Jessell TM, Rafuse VF, Brownstone RM. Functional properties of motoneurons derived from mouse embryonic stem cells. J Neurosci 2004;24:7848-7858.
    22 Pease S, Williams RL. Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor. Exp Cell Res 1990; 190:209-211.
    23 Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271-278.
    24 Owens LV, Xu L, Marston WA, Yang X, Farber MA, Iacocca MV, Cance WG, Keagy BA. Overexpression of the focal adhesion kinase (p125FAK) in the vascular smooth muscle cells of intimal hyperplasia. J Vase Surg 2001;34:344-349.
    25 王建，，赵惠萍，，谢常青．人类胚胎干细胞向造血细胞和内皮细胞诱导分化的初步研究．中国实验血液学杂志 2005；13：222-228．
    26 郭雨霁，高英茂，邴鲁军．无血清培养条件下诱导胚胎干细胞向多巴胺能神经元定向分化的实验研究．解剖学报 2004；35．
    27 汤锋武，李宗金，范存刚，卢士红，刘斌，韩忠朝．小鼠胚胎干细胞在单层粘附培养中向神经细胞的分化．中国生物工程杂志2004；24：78-83．
    1 Thomson JA, Marshall VS, Trojanowski JQ. Neural differentiation of rhesus embryonic stem cells. Apmis 1998;106:149-156; discussion 156-147.
    2 Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 2000;6:88-95.
    3 Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399-404.
    4 Hirose M, Hashimoto H, Shintani N, Nakanishi M, Arakawa N, Iga J, Niwa H, Miyazaki J, Baba A. Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells. Regul Pept 2005; 126:109-113.
    5 Schulz TC, Noggle SA, Palmarini GM, Weiler DA, Lyons IG, Pensa KA, Meedeniya AC, Davidson BP, Lambert NA, Condie BG. Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. Stem Cells 2004;22:1218-1238.
    6 Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19:1134-1140.
    7 Zhang X, Li X, Wu J, Wang Z, Xu H, Yang D. [Isolation, cultivation and identification of stem cells from cerebral cortex of mouse embryo]. Zhonghua Yi Xue Za Zhi 2002;82:832-835.
    8 Passier R, Oostwaard DW, Snapper J, Kloots J, Hassink RJ, Kuijk E, Roelen B, de la Riviere AB, Mummery C. Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures. Stem Cells 2005;23:772-780.
    9 Lavon N, Yanuka O, Benvenisty N. Differentiation and isolation of hepatic-like cells from human embryonic stem cells. Differentiation 2004;72:230-238.
    10 Brolen GK, Heins N, Edsbagge J, Semb H. Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing beta-cell-like cells. Diabetes 2005;54:2867-2874.
    11 Assady S, Maor G, Amit M, Itskovitz-EIdor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001;50:1691-1697.
    12 Zambidis ET, Peault B, Park TS, Bunz F, Civin CI. Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development. Blood 2005;106:860-870.
    13 Qiu C, Hanson E, Olivier E, Inada M, Kaufman DS, Gupta S, Bouhassira EE. Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development. Exp Hematol 2005;33:1450-1458.
    14 Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y. Induction of midbrain dopaminergic neurons from es cells by stromal cell-derived inducing activity. Neuron 2000;28:31-40.
    15 Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. Embryonic stem cells express neuronal properties in vitro. Dev Biol 1995;168:342-357.
    16 Kuo MH, Allis CD. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 1998;20:615-626.
    17 Rathjen J, Dunn S, Bettess MD, Rathjen PD. Lineage specific differentiation of pluripotent cells in vitro: a role for extraembryonic cell types. Reprod Fertil Dev 2001;13:15-22.
    18 Zhang H, Saeki K, Kimura A, Saeki K, Nakahara M, Doshi M, Kondo Y, Nakano T, Yuo A. Efficient and repetitive production of hematopoietic and endothelial cells from feeder-free monolayer culture system of primate embryonic stem cells. Biol Reprod 2006;74:295-306.
    19 Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004;84:767-801.
    20 Gittenberger-de Groot AC, DeRuiter MC, Bergwerff M, Poelmann RE. Smooth muscle cell origin and its relation to heterogeneity in development and disease. Arterioscler Thromb Vasc Biol 1999; 19:1589-1594.
    21 Yoshida T, Owens GK. Molecular determinants of vascular smooth muscle cell diversity. Citc Res 2005;96:280-291.
    22 Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, Wang S, Morton CC, McMahon AP, Powers D, Melton DA. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1353-1356.
    23 Heng BC, Kuleshova LL, Bested SM, Liu H, Cao T. The cryopreservation of human embryonic stem cells. Biotechnol Appl Biochem 2005;41:97-104.
    24 Kim SJ, Park JH, Lee JE, Kim JM, Lee JB, Moon SY, Roh SI, Kim CG, Yoon HS. Effects of type Ⅳ collagen and laminin on the cryopreservation of human embryonic stem cells. Stem Cells 2004;22:950-961.
    25 Chen S, Lechleider RJ. Transforming growth factor-beta-induced differentiation of smooth muscle from a neural crest stem cell line. Circ Res 2004;94:1195-1202.
    26 Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971-974.
    27 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-1147.
    28 Dinsmore J, Ratliff J, Deacon T, Pakzaban P, Jacoby D, Galpem W, Isacson O. Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation. Cell Transplant 1996;5:131-143.
    29 Pal R, Khanna A. Role of hepatocyte-like cells in the differentiation of cardiomyocytes from mouse embryonic stem cells. Stem Cells Dev 2005;14:153-161.
    30 Chen Y, Shao JZ, Xiang LX, Guo J, Zhou QJ, Yao X, Dai LC, Lu YL. Cyclic adenosine 3',5'-monophosphate induces differentiation of mouse embryonic stem cells into cardiomyocytes. Cell Biol Int 2006;30:301-307.
    31 Frid MG, Shekhonin BV, Koteliansky VE, Glukhova MA. Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin. Dev Biol 1992; 153:185-193.
    32 Hungerford JE, Owens GK, Argraves WS, Little CD. Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev Biol 1996;178:375-392.
    33 Duband JL, Gimona M, Scatena M, Sartore S, Small JV. Calponin and SM 22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development. Differentiation 1993;55:1-11.
    34 Mitchell JJ, Reynolds SE, Leslie KO, Low RB, Woodcock-Mitchell J. Smooth muscle cell markers in developing rat lung. Am J Respir Cell Mol Biol 1990;3:515-523.
    35 Miano JM, Cserjesi P, Ligon KL, Periasamy M, Olson EN. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res 1994;75:803-812.
    36 Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 1995;75:487-517.
    37 Lesh RE, Somlyo AP, Owens GK, Somlyo AV. Reversible permeabilization. A novel technique for the intracellular introduction of antisense oligodeoxynucleotides into intact smooth muscle. Circ Res 1995;77:220-230.
    38 Kato S, Shanley JR, Fox JC. Serum stimulation, cell-cell interactions, and extracellular matrix independently influence smooth muscle cell phenotype in vitro. Am J Pathol 1996; 149:687-697.
    39 Wright G, Wang S, Bailey G, Reichenbecher V, Wright GL. Effect of retinoic acid on contractile competence of vascular smooth muscle. Am J Physiol 1996;270:H1363-1370.
    40 Axel DI, Frigge A, Dittmann J, Runge H, Spyridopoulos I, Riessen R, Viebahn R, Karsch KR. All-trans retinoic acid regulates proliferation, migration, differentiation, and extracellular matrix turnover of human arterial smooth muscle cells. Cardiovasc Res 2001;49:851-862.
    41 Kosaka C, Sasaguri T, Komiyama Y, Takahashi H. All-trans retinoic acid inhibits vascular smooth muscle cell proliferation targeting multiple genes for cyelins and cyelin-dependent kinases. Hypertens Res 2001;24:579-588.
    42 Bochaton-Piallat ML, Ropraz P, Gabbiani F, Gabbiani G. Phenotypie heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioseler Thromb Vase Biol 1996; 16:815-820.
    43 Ehler E, Jat PS, Noble MD, Citi S, Draeger A. Vascular smooth muscle cells of H-2Kb-tsA58 transgenic mice. Characterization of cell lines with distinct properties. Circulation 1995;92:3289-3296.
    44 Chamley-Campbell JH, Campbell GR. What controls smooth muscle phenotype? Atheroselerosis 1981;40:347-357.
    45 Miano JM, Berk BC. Retinoids: versatile biological response modifiers of vascular smooth muscle phenotype. Circ Res 2000;87:355-362.
    46 Blank RS, Swartz EA, Thompson MM, Olson EN, Owens GK. A retinoic acid-induced clonal cell line derived from multipotential P19 embryonal carcinoma cells expresses smooth muscle characteristics. Circ Res 1995;76:742-749.
    47 Suzuki T, Kim HS, Kurabayashi M, Hamada H, Fujii H, Aikawa M, Watanabe M, Watanabe N, Sakomura Y, Yazaki Y, Nagai R. Preferential differentiation of P19 mouse embryonal carcinoma cells into smooth muscle cells. Use of retinoie acid and antisense against the central nervous system-specific POU transcription factor Brn-2. Circ Res 1996;78:395-404.
    48 Manabe I, Owens GK. Recruitment of serum response factor and hyperacetylation of histones at smooth muscle-specific regulatory regions during differentiation of a novel P19-derived in vitro smooth muscle differentiation system. Circ Res 2001;88:1127-1134.
    49 Drab M, Hailer H, Bychkov R, Erdmann B, Lindschau C, Haase H, Morano I, Luft FC, Wobus AM. From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model. Faseb J 1997;11:905-915.
    50 Lindskog H, Athley E, Larsson E, Lundin S, Hellstrom M, Lindahl P. New Insights to Vascular Smooth Muscle Cell and Pericyte Differentiation of Mouse Embryonic Stem Cells In Vitro. Arterioscler Thromb Vasc Biol 2006.
    51 Jain MK, Layne MD, Watanabe M, Chin MT, Feinberg MW, Sibinga NE, Hsieh CM, Yet SF, Stemple DL, Lee ME. In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells. J Biol Chem 1998;273:5993-5996.
    52 Watanabe M, Layne MD, Hsieh CM, Maemura K, Gray S, Lee ME, Jain MK. Regulation of smooth muscle cell differentiation by AT-rich interaction domain transcription factors Mrf2alpha and Mrf2beta. Circ Res 2002;91:382-389.
    53 Oishi K, Ogawa Y, Gamoh S, Uehida MK. Contractile responses of smooth muscle cells differentiated from rat neural stem cells. J Physiol 2002;540:139-152.
    54 Pachernik J, esner M, Bryja V, Dvorak P, Hampl A. Neural differentiation of mouse embryonic stem cells grown in monolayer. Reprod Nutr Dev 2002;42:317-326.
    55 Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 1998;125:1747-1757.
    56 Ying QL, Stavridis M, Griffiths D, Li M, Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003; 21: 183-186.
    57 Gerrard L, Rodgers L, Cui W. Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells 2005;23:1234-1241.
    1 Miano JM, Berk BC. Retinoids: versatile biological response modifiers of vascular smooth muscle phenotype. Circ Res 2000;87:355-362.
    2 Kosaka C, Sasaguri T, Komiyama Y, Takahashi H. All-trans retinoic acid inhibits vascular smooth muscle cell proliferation targeting multiple genes for cyclins and cyclin-dependent kinases. Hypertens Res 2001;24:579-588.
    3 Honda M, Hamazaki TS, Komazaki S, Kagechika H, Shudo K, Asashima M. RXR agonist enhances the differentiation of cardiomyocytes derived from embryonic stem cells in serum-free conditions. Biochem Biophys Res Commun 2005;333:1334-1340.
    4 Jimenez-Lara AM, Clarke N, AItucci L, Gronemeyer H. Retinoic-acid-induced apoptosis in leukemia cells. Trends Mol Med 2004;10:508-515.
    5 Bain G, Ray WJ, Yao M, Gottlieb DI. Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem Biophys Res Commun 1996;223:691-694.
    6 Fraichard A, Chassande O, Bilbaut G, Dehay C, Savatier P, Samarut J. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J Cell Sci 1995;108(Pt 10): 3181-3188.
    7 Drab M, Haller H, Bychkov R, Erdmann B, Lindschau C, Haase H, Morano I, Luft FC, Wobus AM. From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model. Faseb J 1997; 11: 905-915.
    8 Njar VC. Cytochrome p450 retinoic acid 4-hydroxylase inhibitors: potential agents for cancer therapy. Mini Rev Med Chem 2002;2:261-269.
    9 Kastner P, Mark M, Ghyselinck N, Krezel W, Dupe V, Grondona JM, Chambon P. Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development. Development 1997;124:313-326.
    10 Napoli JL. Interactions of retinoid binding proteins and enzymes in retinoid metabolism. Biochim Biophys Acta 1999; 1440:139-162.
    11 O'Shea KS. Neuronal differentiation of mouse embryonic stem cells: lineage selection and forced differentiation paradigms. Blood Cells Mol Dis 2001;27:705-712.
    12 Okada Y, Shimazaki T, Sobue G, Okano H. Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev Biol 2004;275:124-142.
    13 Pan Y, Chen X, Wang S, Yang S, Bai X, Chi X, Li K, Liu B, Li L. In vitro neuronal differentiation of cultured human embryonic germ cells. Biochem Biophys Res Commun 2005;327:548-556.
    14 Meyer JS, Katz ML, Maruniak JA, Kirk MD. Neural differentiation of mouse embryonic stem cells in vitro and after transplantation into eyes of mutant mice with rapid retinal degeneration. Brain Res 2004; 1014:131-144.
    15 Evans TR, Kaye SB. Retinoids: present role and future potential. Br J Cancer 1999;80:1-8.
    16 Minucci S, Pelicci PG. Retinoid receptors in health and disease: co-regulators and the chromatin connection. Semin Cell Dev Biol 1999; 10:215-225.
    17 Strahl BD, Allis CD. The language of covalent histone modifications. Nature 2000;403:41-45.
    18 Langston AW, Thompson JR, Gudas LJ. Retinoic acid-responsive enhancers located 3' of the Hox A and Hox B homeobox gene clusters. Functional analysis. J Biol Chem 1997;272:2167-2175.
    19 Blank RS, Swartz EA, Thompson MM, Olson EN, Owens GK. A retinoic acid-induced clonal cell line derived from multipotential P19 embryonal carcinoma cells expresses smooth muscle characteristics. Circ Res 1995;76:742-749.
    20 Chen S, Gardner DG. Retinoic acid uses divergent mechanisms to activate or suppress mitogenesis in rat aortic smooth muscle cells. J Clin Invest 1998;102:653-662.
    21 Liu M, lavarone A, Freedman LP. Transcriptional activation of the human p21 (WAF1/CIP1) gene by retinoic acid receptor. Correlation with retinoid induction of U937 cell differentiation. J Biol Chem 1996;271:31723-31728.
    22 Dittman WA, Nelson SC, Greer PK, Horton ET, Palomba ML, McCachren SS. Characterization of thrombomodulin expression in response to retinoic acid and identification of a retinoic acid response element in the human thrombomodulin gene. J Biol Chem 1994;269:16925-16932.
    23 Bulens F, Ibanez-Tallon I, Van Acker P, De Vriese A, Nelles L, Belayew A, Collen D. Retinoic acid induction of human tissue-type plasminogen activator gene expression via a direct repeat element (DR5) located at -7 kilobases. J Biol Chem 1995;270:7167-7175.
    24 Neuville P, Yan Z, Gidlof A, Pepper MS, Hansson GK, Gabbiani G, Sirsjo A. Retinoic acid regulates arterial smooth muscle cell proliferation and phenotypic features in vivo and in vitro through an RARalpha-dependent signaling pathway. Arterioscler Thromb Vasc Biol 1999;19:1430-1436.
    25 Nagy L, Saydak M, Shipley N, Lu S, Basilion JP, Yan ZH, Syka P, Chandraratna RA, Stein JP, Heyman RA, Davies PJ. Identification and characterization of a versatile retinoid response element (retinoic acid receptor response element-retinoid Ⅹ receptor response element) in the mouse tissue transglutaminase gene promoter. J Biol Chem 1996;271:4355-4365.
    26 Chowdhury ZA, Barsigian C, Chalupowicz GD, Bach TL, Garcia-Manero G, Martinez J. Colocalization of tissue transglutaminase and stress fibers in human vascular smooth muscle cells and human umbilical vein endothelial cells. Exp Cell Res 1997;231:38-49.
    27 Vasios G, Mader S, Gold JD, Leid M, Lutz Y, Gaub MP, Chambon P, Gudas L. The late retinoic acid induction of laminin B1 gene transcription involves RAR binding to the responsive element. Embo J 1991;10:1149-1158.
    28 Hailer H, Lindschau C, Quass P, Distler A, Lufi FC. Differentiation of vascular smooth muscle cells and the regulation of protein kinase C-alpha. Circ Res 1995;76:21-29.
    29 Desai DS, Hirai S, Karnes WE, Jr., Niles RM, Ohno S. Cloning and characterization of the murine PKC alpha promoter: identification of a retinoic acid response element. Biochem Biophys Res Commun 1999;263:28-34.
    30 Suzuki T, Kim HS, Kurabayashi M, Hamada H, Fujii H, Aikawa M, Watanabe M, Watanabe N, Sakomura Y, Yazaki Y, Nagai R. Preferential differentiation of P19 mouse embryonal carcinoma cells into smooth muscle cells. Use of retinoic acid and antisense against the central nervous system-specific POU transcription factor Brn-2. Circ Res 1996;78:395-404.
    31 Minamino N, Shoji H, Sugo S, Kangawa K, Matsuo H. Adrenocortical steroids, thyroid hormones and retinoic acid augment the production of adrenomedullin in vascular smooth muscle cells. Biochem Biophys Res Commun 1995;211:686-693.
    32 de Toledo FG, Cheng J, Dousa TP. Retinoic acid and triiodothyronine stimulate ADP-ribosyl cyclase activity in rat vascular smooth muscle cells. Biochem Biophys Res Commun 1997;238:847-850.
    33 Schule R, Rangarajan P, Yang N, Kliewer S, Ransone LJ, Bolado J, Verma IM, Evans RM. Retinoic acid is a negative regulator of AP-1-responsive genes. Proc Natl Acad Sci U S A 1991;88:6092-6096.
    34 James TW, Wagner R, White LA, Zwolak RM, Brinckerhoff CE. Induction of collagenase and stromelysin gene expression by mechanical injury in a vascular smooth muscle-derived cell line. J Cell Physiol 1993;157:426-437.
    35 Salbert G, Fanjul A, Piedrafita FJ, Lu XP, Kim SJ, Tran P, Pfahl M. Retinoic acid receptors and retinoid Ⅹ receptor-alpha down-regulate the transforming growth factor-beta 1 promoter by antagonizing AP-1 activity. Mol Endocrinol 1993;7:1347-1356.
    36 Barstad RM, Hamers MJ, Stephens RW, Sakariassen KS. Retinoic acid reduces induction of monocyte tissue factor and tissue factor/factor Ⅶa-dependent arterial thrombus formation. Blood 1995;86:212-218.
    37 Hirokawa K, O'Shaughnessy KM, Ramrakha P, Wilkins MR. Inhibition of nitric oxide synthesis in vascular smooth muscle by retinoids. Br J Pharmacol 1994; 113:1448-1454.
    38 Yang JY, Koo BS, Kang MK, Rho HW, Sohn HS, Jhee EC, Park JW. Retinoic acid inhibits inducible nitric oxide synthase expression in 3T3-L1 adipocytes. Exp Mol Med 2002;34:353-360.
    39 Nagy L, Thomazy VA, Chandraratna RA, Heyman RA, Davies PJ. Retinoid-regulated expression of BCL-2 and tissue transglutaminase during the differentiation and apoptosis of human myeloid leukemia (HL-60) cells. Leuk Res 1996;20:499-505.
    40 Zhang Y, Dawson MI, Ning Y, Polin L, Parchment RE, Corbett T, Mohamed AN, Feng KC, Farhana L, Rishi AK, Hogge D, Leid M, Peterson V J, Zhang XK, Mohammad R, Lu JS, Willman C, VanBuren E, Biggar S, Edeistein M, Eilender D, Fontana JA. Induction of apoptosis in retinoid-refractory acute myelogenous leukemia by a novel AHPN analog. Blood 2003; 102:3743-3752.
    41 Garattini E, Parrella E, Diomede L, Gianni M, Kalac Y, Merlini L, Simoni D, Zanier R, Ferrara FF, Chiarucci I, Carminati P, Terao M, Pisano C. ST1926, a novel and orally active retinoid-related molecule inducing apoptosis in myeloid leukemia cells: modulation of intracellular calcium homeostasis. Blood 2004; 103:194-207.
    42 Mologni L, Ponzanelli I, Bresciani F, Sardiello G, Bergamaschi D, Gianni M, Reichert U, Rambaldi A, Terao M, Garattini E. The novel synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) causes apoptosis in acute promyelocytic leukemia cells through rapid activation of caspases. Blood 1999;93:1045-1061.
    43 Marchetti P, Zamzami N, Joseph B, Schraen-Maschke S, Mereau-Richard C, Costantini P, Metivier D, Susin SA, Kroemer G, Formstecher P. The novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid can trigger apoptosis through a mitochondrial pathway independent of the nucleus. Cancer Res 1999;59:6257-6266.
    44 Hail N, Jr., Youssef EM, Lotan R. Evidence supporting a role for mitochondriai respiration in apoptosis induction by the synthetic retinoid CD437. Cancer Res 2001;61:6698-6702.
    45 Zang Y, Beard RL, Chandraratna RA, Kang JX. Evidence of a iysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells. Cell Death Differ 2001;8:477-485.
    46 Nagy L, Thomazy VA, Heyman RA, Davies PJ. Retinoid-induced apoptosis in normal and neoplastic tissues. Cell Death Differ 1998; 5: 11-19.
    47 Napoli JL. Retinoic acid biosynthesis and metabolism. Faseb J 1996; 10:993-1001.
    48 Hasler JA. Pharmacogenetics of cytochromes P450. Mol Aspects Med 1999;20:12-24,25-137.
    49 White JA, Guo YD, Baetz K, Beckett-Jones B, Bonasoro J, Hsu KE, Dilworth FJ, Jones G, Petkovich M. Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase. J Biol Chem 1996;271:29922-29927.
    50 MacLean G, Abu-Abed S, Dolle P, Tahayato A, Chambon P, Petkovich M. Cloning of a novel retinoic-acid metabolizing cytochrome P450, Cyp26B1, and comparative expression analysis with Cyp26A1 during early murine development. Mech Dev 2001; 107:195-201.
    51 Taimi M, Helvig C, Wisniewski J, Ramshaw H, White J, Amad M, Korczak B, Petkovich M. A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and all-trans isomers ofretinoic acid. J Biol Chem 2004;279:77-85.
    52 Ganguly R, Puri IK. Mathematical model for the cancer stem cell hypothesis. Cell Prolif 2006;39:3-14.
    53 Abu-Abed S, Dolle P, Metzger D, Beckett B, Chambon P, Petkovich M. The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures. Genes Dev 2001;15:226-240.
    54 Yashiro K, Zhao X, Uehara M, Yamashita K, Nishijima M, Nishino J, Saijoh Y, Sakai Y, Hamada H. Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse limb. Dev Cell 2004; 6: 411-422.
    55 Ozpolat B, Mehta K, Tari AM, Lopez-Berestein G. all-trans-Retinoic acid-induced expression and regulation of retinoic acid 4-hydroxylase (CYP26) in human promyelocytic leukemia. Am J Hematol 2002;70:39-47.
    1 Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;78:7634-7638.
    2 Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. Embryonic stem cells express neuronal properties in vitro. Dev Biol 1995;168:342-357.
    3 Dinsmore J, Ratliff J, Deacon T, Pakzaban P, Jacoby D, Galpern W, Isacson O. Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation. Cell Transplant 1996;5:131-143.
    4 Sakakibara S, lmai T, Hamaguchi K, Okabe M, Aruga J, Nakajima K, Yasutomi D, Nagata T, Kurihara Y, Uesugi S, Miyata T, Ogawa M, Mikoshiba K, Okano H. Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev Biol 1996;176:230-242.
    5 Hirst SJ, Twort CH, Lee TH. Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype. Am J Respir Cell Moi Biol 2000;23:335-344.
    6 Kawasaki H, Suemori H, Mizuseki K, Watanabe K, Urano F, Ichinose H, Haruta M, Takahashi M, Yoshikawa K, Nishikawa S, Nakatsuji N, Sasai Y. 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.
    7 Barberi T, Klivenyi P, Calingasan NY, Lee H, Kawamata H, Loonam K, Perrier AL, Bruses J, Rubio ME, Topf N, Tabar V, Harrison NL, Beal MF, Moore MA, Studer L. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 2003;21: 1200-1207.
    8 Nakayama T, Momoki-Soga T, Inoue N. Astrocyte-derived factors instruct differentiation of embryonic stem cells into neurons. Neurosci Res 2003;46:241-249.
    9 Blaheta RA, Cinatl J, Jr. Anti-tumor mechanisms of valproate: a novel role for an old drug. Med Res Rev 2002;22:492-511.
    10 Woo HJ, Lee SJ, Choi BT, Park YM, Choi YH. Induction of apoptosis and inhibition of telomerase activity by trichostatin A, a histone deacetylase inhibitor, in human leukemic U937 cells. Exp Mol Pathol 2006.
    11 Hsieh J, Nakashima K, Kuwabara T, Mejia E, Gage FH. Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc Natl Acad Sci U S A 2004; 101: 16659-16664.
    12 DeLorenzo RJ, Pal S, Sombati S. Prolonged activation of the N-methyl-D-aspartate receptor-Ca2+ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture. Proc Natl Acad Sci U S A 1998;95:14482-14487.
    13 Yamitsky D, Gross Y, Lorian A, Shalev A, Lamensdorf I, Bornstein R, Shorer S, Mayevsky A, Patel KP, Abbott NJ, Mayhan WG. Blood-brain barrier opened by stimulation of the parasympathetic sphenopalatine ganglion: a new method for macromolecule delivery to the brain. J Neurosurg 2004; 101: 303-309.
    14 Spencer VA, Davie JR. Role of covalent modifications of histones in regulating gene expression. Gene 1999;240:1-12.
    15 Pogo BG, Allfrey VG, Mirsky AE. RNA synthesis and histone acetylation during the course of gene activation in lymphocytes. Proc Natl Acad Sci U S A 1966;55:805-812.
    16 Dokmanovic M, Marks PA. Prospects: histone deacetylase inhibitors. J Cell Biochem 2005;96:293-304.
    17 Kuo MH, Allis CD. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 1998;20:615-626.
    18 Cress WD, Seto E. Histone deacetylases, transcriptional control, and cancer. J Cell Physiol 2000;184:1-16.
    19 Richon VM, O'Brien JP. Histone deacetylase inhibitors: a new class of potential therapeutic agents for cancer treatment. Clin Cancer Res 2002;8:662-664.
    20 Moradei O, Maroun CR, Paquin I, Vaisburg A. Histone deacetylase inhibitors: latest developments, trends and prospects. Curr Med Chem Anticancer Agents 2005;5:529-560.
    21 Hess-Stumpp H. Histone deacetylase inhibitors and cancer: from cell biology to the clinic. Ear J Cell Biol 2005;84:109-121.
    22 Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature 2005;434:843-850.
    23 Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105-111.
    24 Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke ME Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100:3983-3988.
    25 Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB. Identification of a cancer stem cell in human brain tumors. Cancer Res 2003;63:5821-5828.
    26 Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730-737.
    27 Ganguly R, Puri IK. Mathematical model for the cancer stem cell hypothesis. Cell Prolif 2006;39:3-14.
    28 Szutorisz H, Canzonetta C, Georgiou A, Chow CM, Tora L, Dillon N. Formation of an active tissue-specific chromatin domain initiated by epigenetic marking at the embryonic stem cell stage. Mol Cell Biol 2005;25:1804-1820.
    29 Meshorer E, Misteli T. Chromatin in pluripotent embryonic stem cells and differentiation. Nat Rev Mol Cell Biol 2006;7:540-546.
    30 Kaji K, Caballero IM, MacLeod R, Nichols J, Wilson VA, Hendrich B. The NuRD component Mbd3 is required for pluripotency of embryonic stem cells. Nat Cell Biol 2006;8:285-292.
    31 Rambhatla L, Chiu CP, Kundu P, Peng Y, Carpenter MK. Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant 2003;12:1-11.
    32 周庆军．胚胎干细胞体外分化为肝细胞的实验研究．[博士学位论文]．杭州：浙江大学，2006．
    33 Illi B, Scopece A, Nanni S, Farsetti A, Morgante L, Biglioli P, Capogrossi MC, Gaetano C. Epigenetic histone modification and cardiovascular lineage programming in mouse embryonic stem cells exposed to laminar shear stress. Circ Res 2005;96:501-508.
    34 Zandonella C. Stem-cell therapies: the first wave. Nature 2005;435:877-878.
    35 van Inzen WG, Peppelenbosch MP, van den Brand MW, Tertoolen LG, de Laat SW. Neuronal differentiation of embryonic stem cells. Biochim Biophys Acta 1996; 1312:21-26.
    36 Tropepe V, Hitoshi S, Sirard C, Mak TW, Rossant J, van der Kooy D. Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 2001;30:65-78.
    37 Ying QL, Stavridis M, Griffiths D, Li M, Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003;21: 183-186.