中文摘要
間質幹細胞（Mesenchymal stem cell)為成體幹細胞的一種，主要存在於骨髓、週邊血臍帶、胎盤、或者脂肪組織中。間質幹細胞為一種具多功能性之幹細胞，可以分化為脂肪、硬骨、軟骨、肌肉、神經等細胞，因此在間質幹細胞在再生醫學或組織工程領域成為極具潛力之研究主題，亦廣泛應用於傷害修補與再生。一般而言，間質幹細胞之分化往往透過給予適當之生長激素誘導分化，不但花費昂貴，且所需時間較長。本研究中嘗試給予間質幹細胞物理性刺激，藉由改變細胞骨架或給予胞外基質的培養方式探討對間葉幹細胞分化之影響。首先利用ROCK抑制劑Y-27632研究RhoA激酶對於胎盤幹細胞分化之影響。研究發現透過Y-27632處理後，胎盤幹細胞產生型態改變，表現像神經細胞的型態，並能表現神經前趨細胞標誌蛋白。與傳統視黃酸，(Retinoic Acid)誘導方式相比，抑制RhoA激酶能更快速地引發神經分化，並且不影響胎盤幹細胞的正常生長曲線及細胞週期。研究更近一步確認，針對RhoA激酶做專一性減弱 （Knockdown) 或對及其下游途徑之肌球蛋白輕鍊激酶（MLCK) 做抑制， 皆可得到相似的結果。這些研究結果顯示RhoA-RhoA激酶訊號途徑之抑制有助於胎盤幹細胞向神經譜系分化之傾向；此外，較輕微的生長及細胞週期影響也有利於未來胎盤幹細胞分化及應床臨用之參考。第二部分則是使用了骨頭衍生之間質幹細胞，研究發現當給與了細胞外基質時，可以使得間葉幹細胞於四小時內快速分化為內皮細胞。間葉幹細胞透過胞外基質處理後，不只表現內皮細胞的標誌蛋白，也能形成如血管般的環狀構造，並能夠如一般表皮細胞一樣攝取低密度脂蛋白。另外在體內實驗中，也能如同人類臍帶靜脈內皮細胞一樣促進雞胚絨毛膜上的血管新生。進一步地研究發現胞外基質誘使間質幹細胞中的轉譯因子FOXC2大量表現。透過專一性的抑制，我們認為FOXC2在胞外基質誘導之內皮分化扮演了最重要的角色，FOXC2不只調節內皮細胞的標誌蛋白之表現，也調控下游基因integrin avb3/CD61進而影響血管新生。除此之外，我們亦分析胞外基質的主要成份，發現胞外基質中的Laminin對於FOXC2的表現影響最大。
綜言之，本研究透過改變細胞骨架與胞外基質誘導間質幹細胞之分化，結果顯示當給予上述刺激後，間質幹細胞能夠快速地走向神經或內皮細胞而不影響其生長，此研究成果可為未來間質幹細胞臨床應用之參考。

Abstract
Mesenchymal stem cells (MSCs) are one kind of adult stem cells which can be isolated from diverse sources of tissue, including bone marrow, peripheral blood, umbilical cord, placenta, and adipose tissue. MSCs are multipotent stem cell that can differentiate into various mesodermal cell types, including adipocytes, osteoblasts, chondrocytes, muscle cells, or even ectodermal and endodermal cell types such as neurons and hepatocytes, respectively. Therefore, MSCs have become good potential therapeutic candidates for use in tissue engineering and regenerative medicine. Differentiation of MSCs is usually achieved by using growth factors; however, the procedure is expensive and requires longer periods of time for differentiation. Here, we investigate the effects of manipulating biophysical parameters on MSC differentiation, including alteration of the intracellular cytoskeleton and the extracellular matrix (ECM)/external microenvironment of the stem cell. In the first part, we report on the efficient differentiation of placental-derived multipotent cells (PDMCs)—a type of MSC isolated from human term placenta—into a neural phenotype with use of Y-27632, a clinically compliant small molecular inhibitor of Rho kinase (ROCK) which is a major mediator of the intracellular actin-myosin cytoskeleton. Y-27632 induces a higher percentage of neural-like cells in PDMCs without arresting proliferation or cell cycle dynamics. Y-27632-treated PDMCs express several neural lineage genes at the RNA and protein level, including nestin, MAP2, and GFAP. The effect of the ROCK inhibitor is cell-specific to PDMCs, and is mainly mediated through the ROCK2 isoform and its downstream target, myosin II. In the second part, we investigated the endothelial differentiation capacity of MSCs derived from bone tissue (B-MSC) as mediated by manipulating the extracellular microenviroment in terms of the ECM. We found that fetal pre-osteoblast and adult trabecular bone derived (TB) MSCs cultured in ECM can not only enhance endothelial specific marker expression within 4 hours treatment but also form tubular structures and uptake acetylated low-density lipoproteins, fulfilling the functional criteria for endothelial cells. Moreover, addition of B-MSCs but not other cells significantly enhanced vessel formation in the in vivo chick chorioallantoic membrane assay. Mechanistically, this appears to be due to the upregulation of the endothelial transcription factor forkhead box protein C2 (FOXC2) and its downstream gene v3 integrin/CD61in B-MSCs but not BMMSCs by laminin, a component protein of the ECM.
Taken together, our data suggest that manipulation of biophysical parameters of MSCs can influence differentiation capacity, as seen in the rapid induction of a neural or endothelial phenotype with the alteration of cytoskeleton or changing the external culturing environment, respectively. These results provide evidence for using non-biological methods—which are robust and may be more cost-effective—to manipulate MSCs and other stem cells in therapeutic use for tissue engineering and regenerative medicine.

Reference
[1] Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145-7.
[2] 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.
[3] Baldwin T. Morality and human embryo research. Introduction to the Talking Point on morality and human embryo research. EMBO Rep. 2009;10:299-300.
[4] Solter D. From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research. Nat Rev Genet. 2006;7:319-27.
[5] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663-76.
[6] Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, et al. Disease-specific induced pluripotent stem cells. Cell. 2008;134:877-86.
[7] Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther. 2003;5:32-45.
[8] Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity. 1994;1:661-73.
[9] Gage FH. Mammalian neural stem cells. Science. 2000;287:1433-8.
[10] Webster C, Pavlath GK, Parks DR, Walsh FS, Blau HM. Isolation of human myoblasts with the fluorescence-activated cell sorter. Exp Cell Res. 1988;174:252-65.
[11] Thomas ED, Lochte HL, Jr., Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med. 1957;257:491-6.
[12] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143-7.
[13] Lu LL, Liu YJ, Yang SG, Zhao QJ, Wang X, Gong W, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica. 2006;91:1017-26.
[14] Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211-28.
[15] Yen BL, Huang HI, Chien CC, Jui HY, Ko BS, Yao M, et al. Isolation of multipotent cells from human term placenta. Stem Cells. 2005;23:3-9.
[16] Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res. 2000;2:477-88.
[17] Brighton CT, Lorich DG, Kupcha R, Reilly TM, Jones AR, Woodbury RA, 2nd. The pericyte as a possible osteoblast progenitor cell. Clin Orthop Relat Res. 1992:287-99.
[18] Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A. 1999;96:10711-6.
[19] Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells. 2003;21:105-10.
[20] Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell. 2008;3:301-13.
[21] Li H, Yu B, Zhang Y, Pan Z, Xu W, Li H. Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes. Biochem Biophys Res Commun. 2006;341:320-5.
[22] Oswald J, Boxberger S, Jorgensen B, Feldmann S, Ehninger G, Bornhauser M, et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells. 2004;22:377-84.
[23] Muguruma Y, Yahata T, Miyatake H, Sato T, Uno T, Itoh J, et al. Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood. 2006;107:1878-87.
[24] Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol. 2006;6:93-106.
[25] Kiel MJ, Morrison SJ. Uncertainty in the niches that maintain haematopoietic stem cells. Nat Rev Immunol. 2008;8:290-301.
[26] Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99:3838-43.
[27] Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 2002;30:42-8.
[28] Kadiyala S, Young RG, Thiede MA, Bruder SP. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant. 1997;6:125-34.
[29] Richards M, Huibregtse BA, Caplan AI, Goulet JA, Goldstein SA. Marrow-derived progenitor cell injections enhance new bone formation during distraction. J Orthop Res. 1999;17:900-8.
[30] Johnstone B, Yoo JU. Autologous mesenchymal progenitor cells in articular cartilage repair. Clin Orthop Relat Res. 1999:S156-62.
[31] Young RG, Butler DL, Weber W, Caplan AI, Gordon SL, Fink DJ. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res. 1998;16:406-13.
[32] Neubauer M, Hacker M, Bauer-Kreisel P, Weiser B, Fischbach C, Schulz MB, et al. Adipose tissue engineering based on mesenchymal stem cells and basic fibroblast growth factor in vitro. Tissue Eng. 2005;11:1840-51.
[33] Choi YS, Park SN, Suh H. Adipose tissue engineering using mesenchymal stem cells attached to injectable PLGA spheres. Biomaterials. 2005;26:5855-63.
[34] Sottile J. Regulation of angiogenesis by extracellular matrix. Biochim Biophys Acta. 2004;1654:13-22.
[35] Inoue K, Ohgushi H, Yoshikawa T, Okumura M, Sempuku T, Tamai S, et al. The effect of aging on bone formation in porous hydroxyapatite: biochemical and histological analysis. J Bone Miner Res. 1997;12:989-94.
[36] Mueller SM, Glowacki J. Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges. J Cell Biochem. 2001;82:583-90.
[37] Rao MS, Mattson MP. Stem cells and aging: expanding the possibilities. Mech Ageing Dev. 2001;122:713-34.
[38] Yourek G, McCormick SM, Mao JJ, Reilly GC. Shear stress induces osteogenic differentiation of human mesenchymal stem cells. Regen Med. 2010;5:713-24.
[39] Connelly JT, Vanderploeg EJ, Mouw JK, Wilson CG, Levenston ME. Tensile loading modulates bone marrow stromal cell differentiation and the development of engineered fibrocartilage constructs. Tissue Eng Part A. 2010;16:1913-23.
[40] Evans ND, Minelli C, Gentleman E, LaPointe V, Patankar SN, Kallivretaki M, et al. Substrate stiffness affects early differentiation events in embryonic stem cells. Eur Cell Mater. 2009;18:1-13; discussion -4.
[41] Philp D, Chen SS, Fitzgerald W, Orenstein J, Margolis L, Kleinman HK. Complex extracellular matrices promote tissue-specific stem cell differentiation. Stem Cells. 2005;23:288-96.
[42] Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res. 2002;69:880-93.
[43] Ninomiya Y, Adams R, Morriss-Kay GM, Eto K. Apoptotic cell death in neuronal differentiation of P19 EC cells: cell death follows reentry into S phase. J Cell Physiol. 1997;172:25-35.
[44] Liao JK, Seto M, Noma K. Rho kinase (ROCK) inhibitors. J Cardiovasc Pharmacol. 2007;50:17-24.
[45] Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 2008;132:631-44.
[46] Grellier M, Bordenave L, Amedee J. Cell-to-cell communication between osteogenic and endothelial lineages: implications for tissue engineering. Trends Biotechnol. 2009;27:562-71.
[47] Grellier M, Ferreira-Tojais N, Bourget C, Bareille R, Guillemot F, Amedee J. Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells. J Cell Biochem. 2009;106:390-8.
[48] Unger RE, Sartoris A, Peters K, Motta A, Migliaresi C, Kunkel M, et al. Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials. Biomaterials. 2007;28:3965-76.
[49] Rouwkema J, de Boer J, Van Blitterswijk CA. Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct. Tissue Eng. 2006;12:2685-93.
[50] Ostenfeld T, Svendsen CN. Recent advances in stem cell neurobiology. Adv Tech Stand Neurosurg. 2003;28:3-89.
[51] Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000;164:247-56.
[52] Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000;61:364-70.
[53] Yen BL, Chien CC, Chen YC, Chen JT, Huang JS, Lee FK, et al. Placenta-derived multipotent cells differentiate into neuronal and glial cells in vitro. Tissue Eng Part A. 2008;14:9-17.
[54] Chang CJ, Yen ML, Chen YC, Chien CC, Huang HI, Bai CH, et al. Placenta-derived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-gamma. Stem Cells. 2006;24:2466-77.
[55] Liu KJ, Wang CJ, Chang CJ, Hu HI, Hsu PJ, Wu YC, et al. Surface expression of HLA-G is involved in mediating immunomodulatory effects of placenta-derived multipotent cells (PDMCs) towards natural killer lymphocytes. Cell Transplant. 2011;10-11:1721-30.
[56] Brooke G, Cook M, Blair C, Han R, Heazlewood C, Jones B, et al. Therapeutic applications of mesenchymal stromal cells. Semin Cell Dev Biol. 2007;18:846-58.
[57] McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell. 2004;6:483-95.
[58] Pacary E, Legros H, Valable S, Duchatelle P, Lecocq M, Petit E, et al. Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells. J Cell Sci. 2006;119:2667-78.
[59] Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003;4:446-56.
[60] Strauss S. Geron trial resumes, but standards for stem cell trials remain elusive. Nature Biotechnology. 2010;28:989-90.
[61] Keirstead HS. Stem cell transplantation into the central nervous system and the control of differentiation. Journal of Neuroscience Research. 2001;63:233-6.
[62] Neal BS, Sparber SB. Long-term effects of neonatal exposure to isobutylmethylxanthine. I. Retardation of learning with antagonism by mianserin. Psychopharmacology (Berl). 1991;103:388-97.
[63] Liu Y, Song Z, Zhao Y, Qin H, Cai J, Zhang H, et al. A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells. Biochem Biophys Res Commun. 2006;346:131-9.
[64] Schwartz PH, Brick DJ, Stover AE, Loring JF, Muller FJ. Differentiation of neural lineage cells from human pluripotent stem cells. Methods. 2008;45:142-58.
[65] Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998;279:509-14.
[66] Luo L. Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci. 2000;1:173-80.
[67] Carvalho RS, Schaffer JL, Gerstenfeld LC. Osteoblasts induce osteopontin expression in response to attachment on fibronectin: demonstration of a common role for integrin receptors in the signal transduction processes of cell attachment and mechanical stimulation. J Cell Biochem. 1998;70:376-90.
[68] Spiegelman BM, Ginty CA. Fibronectin modulation of cell shape and lipogenic gene expression in 3T3-adipocytes. Cell. 1983;35:657-66.
[69] Chklovskii DB. Synaptic connectivity and neuronal morphology: two sides of the same coin. Neuron. 2004;43:609-17.
[70] Da Silva JS, Medina M, Zuliani C, Di Nardo A, Witke W, Dotti CG. RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability. J Cell Biol. 2003;162:1267-79.
[71] Lehmann M, Fournier A, Selles-Navarro I, Dergham P, Sebok A, Leclerc N, et al. Inactivation of Rho signaling pathway promotes CNS axon regeneration. J Neurosci. 1999;19:7537-47.
[72] Pacary E, Tixier E, Coulet F, Roussel S, Petit E, Bernaudin M. Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth. Mol Cell Neurosci. 2007;35:409-23.
[73] Lawrence DA, Colinas RJ, Walsh AC. Influence of oxygen partial pressure on human and mouse myeloid cell line characteristics. Fundam Appl Toxicol. 1996;29:287-93.
[74] Chang TC, Chen YC, Yang MH, Chen CH, Hsing EW, Ko BS, et al. Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cell plating density-dependent manner. PLoS One. 2012;5:e9187.
[75] Kitani H, Ikeda H, Atsumi T, Watanabe R. Efficiency of neural differentiation of mouse P19 embryonal carcinoma cells is dependent on the seeding density. Cell Transplant. 1997;6:521-5.
[76] Lu H, Guo L, Wozniak MJ, Kawazoe N, Tateishi T, Zhang X, et al. Effect of cell density on adipogenic differentiation of mesenchymal stem cells. Biochem Biophys Res Commun. 2009;381:322-7.
[77] Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 2000;57:976-83.
[78] Lock FE, Hotchin NA. Distinct roles for ROCK1 and ROCK2 in the regulation of keratinocyte differentiation. PLoS One. 2009;4:e8190.
[79] Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. European cells & materials. 2008;15:100-14.
[80] Carano RA, Filvaroff EH. Angiogenesis and bone repair. Drug discovery today. 2003;8:980-9.
[81] Gerber HP, Ferrara N. Angiogenesis and bone growth. Trends in cardiovascular medicine. 2000;10:223-8.
[82] Kronenberg HM. Developmental regulation of the growth plate. Nature. 2003;423:332-6.
[83] Rouwkema J, Rivron NC, van Blitterswijk CA. Vascularization in tissue engineering. Trends in biotechnology. 2008;26:434-41.
[84] Santos MI, Reis RL. Vascularization in bone tissue engineering: physiology, current strategies, major hurdles and future challenges. Macromolecular bioscience. 2010;10:12-27.
[85] Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 1997;89:747-54.
[86] Marie PJ. Transcription factors controlling osteoblastogenesis. Archives of biochemistry and biophysics. 2008;473:98-105.
[87] Owen M, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp. 1988;136:42-60.
[88] Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276:71-4.
[89] Noth U, Osyczka AM, Tuli R, Hickok NJ, Danielson KG, Tuan RS. Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. J Orthop Res. 2002;20:1060-9.
[90] Sakaguchi Y, Sekiya I, Yagishita K, Ichinose S, Shinomiya K, Muneta T. Suspended cells from trabecular bone by collagenase digestion become virtually identical to mesenchymal stem cells obtained from marrow aspirates. Blood. 2004;104:2728-35.
[91] Yen ML, Chien CC, Chiu IM, Huang HI, Chen YC, Hu HI, et al. Multilineage differentiation and characterization of the human fetal osteoblastic 1.19 cell line: a possible in vitro model of human mesenchymal progenitors. Stem Cells. 2007;25:125-31.
[92] Grellier M, Bordenave L, Amedee J. Cell-to-cell communication between osteogenic and endothelial lineages: implications for tissue engineering. Trends in biotechnology. 2009;27:562-71.
[93] Grellier M, Ferreira-Tojais N, Bourget C, Bareille R, Guillemot F, Amedee J. Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells. Journal of cellular biochemistry. 2009;106:390-8.
[94] Guillotin B, Bareille R, Bourget C, Bordenave L, Amedee J. Interaction between human umbilical vein endothelial cells and human osteoprogenitors triggers pleiotropic effect that may support osteoblastic function. Bone. 2008;42:1080-91.
[95] Villars F, Guillotin B, Amedee T, Dutoya S, Bordenave L, Bareille R, et al. Effect of HUVEC on human osteoprogenitor cell differentiation needs heterotypic gap junction communication. Am J Physiol Cell Physiol. 2002;282:C775-85.
[96] Rouwkema J, de Boer J, Van Blitterswijk CA. Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct. Tissue engineering. 2006;12:2685-93.
[97] Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L, et al. A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell. 1997;89:981-90.
[98] Janeczek Portalska K, Leferink A, Groen N, Fernandes H, Moroni L, van Blitterswijk C, et al. Endothelial differentiation of mesenchymal stromal cells. PLoS One. 2012;7:e46842.
[99] Oskowitz A, McFerrin H, Gutschow M, Carter ML, Pochampally R. Serum-deprived human multipotent mesenchymal stromal cells (MSCs) are highly angiogenic. Stem Cell Res. 2011;6:215-25.
[100] Wang H, Riha GM, Yan S, Li M, Chai H, Yang H, et al. Shear stress induces endothelial differentiation from a murine embryonic mesenchymal progenitor cell line. Arterioscler Thromb Vasc Biol. 2005;25:1817-23.
[101] Wu CC, Chao YC, Chen CN, Chien S, Chen YC, Chien CC, et al. Synergism of biochemical and mechanical stimuli in the differentiation of human placenta-derived multipotent cells into endothelial cells. J Biomech. 2008;41:813-21.
[102] Su JL, Chiou J, Tang CH, Zhao M, Tsai CH, Chen PS, et al. CYR61 regulates BMP-2-dependent osteoblast differentiation through the {alpha}v{beta}3 integrin/integrin-linked kinase/ERK pathway. The Journal of biological chemistry. 2010;285:31325-36.
[103] Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. Angiogenesis assays: a critical overview. Clin Chem. 2003;49:32-40.
[104] De Luca A, Gallo M, Aldinucci D, Ribatti D, Lamura L, D'Alessio A, et al. Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells. Journal of Cellular Physiology. 2011;226:2131-8.
[105] Tseng PC, Young TH, Wang TM, Peng HW, Hou SM, Yen ML. Spontaneous osteogenesis of MSCs cultured on 3D microcarriers through alteration of cytoskeletal tension. Biomaterials. 2012;33:556-64.
[106] Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315-7.
[107] De Val S, Black BL. Transcriptional control of endothelial cell development. Developmental cell. 2009;16:180-95.
[108] De Val S, Chi NC, Meadows SM, Minovitsky S, Anderson JP, Harris IS, et al. Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors. Cell. 2008;135:1053-64.
[109] Hayashi H, Sano H, Seo S, Kume T. The Foxc2 transcription factor regulates angiogenesis via induction of integrin beta3 expression. The Journal of biological chemistry. 2008;283:23791-800.
[110] Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, Martin GR. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982;21:6188-93.
[111] Ducy P, Schinke T, Karsenty G. The osteoblast: a sophisticated fibroblast under central surveillance. Science. 2000;289:1501-4.
[112] Takada I, Suzawa M, Matsumoto K, Kato S. Suppression of PPAR transactivation switches cell fate of bone marrow stem cells from adipocytes into osteoblasts. Ann N Y Acad Sci. 2007;1116:182-95.
[113] Wright E, Hargrave MR, Christiansen J, Cooper L, Kun J, Evans T, et al. The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet. 1995;9:15-20.
[114] Ferdous A, Caprioli A, Iacovino M, Martin CM, Morris J, Richardson JA, et al. Nkx2-5 transactivates the Ets-related protein 71 gene and specifies an endothelial/endocardial fate in the developing embryo. Proc Natl Acad Sci U S A. 2009;106:814-9.
[115] Kume T, Jiang H, Topczewska JM, Hogan BL. The murine winged helix transcription factors, Foxc1 and Foxc2, are both required for cardiovascular development and somitogenesis. Genes & development. 2001;15:2470-82.
[116] Davis GE, Senger DR. Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ Res. 2005;97:1093-107.
[117] Mahabeleshwar GH, Feng W, Phillips DR, Byzova TV. Integrin signaling is critical for pathological angiogenesis. The Journal of experimental medicine. 2006;203:2495-507.
[118] Bader BL, Rayburn H, Crowley D, Hynes RO. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins. Cell. 1998;95:507-19.
[119] Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science. 1994;264:569-71.
[120] Schneller M, Vuori K, Ruoslahti E. Alphavbeta3 integrin associates with activated insulin and PDGFbeta receptors and potentiates the biological activity of PDGF. EMBO J. 1997;16:5600-7.
[121] Soldi R, Mitola S, Strasly M, Defilippi P, Tarone G, Bussolino F. Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J. 1999;18:882-92.
[122] Gonzalez AM, Gonzales M, Herron GS, Nagavarapu U, Hopkinson SB, Tsuruta D, et al. Complex interactions between the laminin alpha 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo. Proc Natl Acad Sci U S A. 2002;99:16075-80.
[123] Estrach S, Cailleteau L, Franco CA, Gerhardt H, Stefani C, Lemichez E, et al. Laminin-binding integrins induce Dll4 expression and Notch signaling in endothelial cells. Circ Res. 2011;109:172-82.
[124] Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res. 2004;95:343-53.
[125] Krenning G, van Luyn MJ, Harmsen MC. Endothelial progenitor cell-based neovascularization: implications for therapy. Trends Mol Med. 2009;15:180-9.