本研究是以小鼠胚幹細胞作為實驗的細胞種類，分別在二維培養及三維培養系統中，探討不同培養基成分誘導幹細胞分化的潛力，並且評估微環境對幹細胞分化所產生的影響。
在二維培養的情況下，利用多種不同的細胞培養基作為D3 cell line的養分來源，包含D3 cell culture medium (DMEM with 15% FBS) only，D3 cell culture medium with 0.5M cyclic AMP and 10mM retionic acid，D3 cell culture medium with 20mM retionic acid，D3 cell culture medium with 20mM Ca++，M199 culture medium with 10 ng/ml bFGF，Keratinocyte growth medium等，評估不同成分對於幹細胞分化為內皮細胞、表皮細胞與神經細胞的影響。除此之外，我們改變細胞培養的條件，如在培養盤表面塗布一層膠原蛋白，以探討微環境對幹細胞生長與分化造成的效應。研究結果顯示在D3 cell culture medium only培養情況下，我們可得到較多數目的內皮細胞。
在三維培養的情況中，以磁珠分離的方式獲得在二維培養下已分化之內皮細胞，並且利用本實驗室所研發之豬源多孔性膠原蛋白基質做為培養材料，進一步將人類纖維母細胞與內皮細胞共同培養在多孔性膠原蛋白基質中。透過免疫組織化學染色的方式，我們觀察到有類微血管結構的生成，以及發現內皮細胞會表達出其分子標誌CD34與vWF。其所形成的管狀結構周圍，亦有明顯的膠原蛋白第四型分子的生成。

Using the mouse embryonic stem cells (ES cells), we demonstrated the effects of culture medium with different supplements on induction potentials in 2-D and 3-D cultural systems and also evaluated the influences of microenvironments on cell differentiation.
In two dimensional cultivation, we used several culture mediums to be the nutrient supply including D3 cell culture medium (DMEM with 15%FBS) only, D3 cell culture medium with 0.5M cyclic AMP and 10mM Retionic acid, D3 cell culture medium with 20mM retionic acid, D3 cell culture medium with 20mM Ca++, M199 culture medium with 10ng/ml bFGF, and Keratinocyte growth medium etc., to evaluate the influences on the differentiation of stem cells into endothelial cells, keratinocytes and neurons. Besides that, in order to study the effects of microenvironments on the growth and differentiation of ES cells, we changed the microenvironments on cell cultural conditions, such as coating collagen on the culture plate surface. The Results indicated that we obtained more endothelial cells by using D3 culture medium only.
In three dimensional cultivations, we separated endothelial cells by the way of magnetic beads from differentiated cell populations and used porcine porous collagen matrices as cultural materials, then co-cultured human fibroblasts with endothelial cells in the matrices. Through the immunohistochemical staining, we observed that the micro capillary-like tube formation and found that endothelial cells expressed CD34 and vWF, the molecular markers of endothelial cells. In the tube surroundings, there were also significant signals of type IV collagen depositions.

Alison, M.R., Poulsom, R., Jeffery, R., Dhillon, A.P., Quaglia, A., Jacob, J., Novelli, M., Prentice, G., Williamson, J., and Wright, N.A. Hepatocytes from non-hepatic adult stem cells. Nature 406, 257. 2000
Anderson, D.J., Gage, F.H., and Weissman, I.L. Can stem cells cross lineage boundaries? Nat. Med. 7, 393-395. 2001
Bain, G., Kitchens, D., Yao, M., Huettner, J.E., and Gottlieb, D.I. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342-357. 1995
Bagutti, C., Wobus, A.M., Fassler, R., and Watt, F.M. Differentiation of embryonal stem cells into keratinocytes: comparison of wild-type and β(1) integrin-deficient cells. Dev. Biol. 179, 184-196. 1996
Baylis F. Human embryonic stem cell lines: the ethics of derivation. J Obstet Gynaecol Can. Feb;24(2):159-63. 2002
Bjornson, C.R., Rietze, R.L., Reynolds, B.A., Magli, M.C., and Vescovi, A.L. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cell in vivo. Science 283, 534-537. 1999
Brazelton, T.R., Rossi, F.M., Keshet, G.I., and Blau, H.M. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290, 1775-1779. 2000
Brustle, O., Jones, K.N., Learish, R.D., Karram, K., Choudhary, K., Wiestler, O.D., Duncan, I.D., and McKay, R.D. Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science. 285, 754-756. 1999
Clarke, D.L., Johansson, C.B., Wilbertz, J., Veress, B., Nilsson, E., Karlstrom, H., Lendahl, U., and Frisen, J. Generation potential of adult neuronal stem cells. Science 288, 1660-1663, 2000
Dani, C., Smith, A.G., Dessolin, S., Leroy, P., Staccini, L., Villageois, P., Darimont, C., and Ailhaud, G. Differentiation of embryonic stem cells into adipocytes in vitro. J. Cel. Sci. 110, 1279-1285. 1997
Daniel, R.M., Richard, L.G., David, G. Stem cell biology. Cold spring harbor laboratory press, Cold Spring Harbor, New York. 2001
Doerflinger RM. The ethics of funding embryonic stem cell research: a Catholic viewpoint. Kennedy Inst Ethics J. Jun;9(2):137-50, 1999
Doetschman, T., Eistetter, H., Katz, M., Schmit, W., and Kemler, R. The in vitro development of blastocystderived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morph. 87, 27-45. 1985
Eiges, R., Schuldiner, M., Drukker, M., Yanuka, O., Itskovitz-Eldor, J., and Benvenisty, N. Establishment of human embryonic stem cell-transduced clones carrying a marker of undifferentiated cells. Curr. Biol. 11, 514-518, 2001
Fairchild, P.J., Brook, F.A., Gardner, R.L., Graca, L., Strong, V., Tone, Y., Tone, M., Nolan, K.F., and Waldmann, H. Directed differentiation of dendritic cells from mouse embryonic stem cells. Curr. Biol. 10, 1515-1518. 2000
Fernandez Lamelas MA, Garcia-Mayor R. Embryonic stem cell research: the relevance of ethics in the progress of science and the moral and ethical quandry of embryonic stem cell research. Med Sci Monit. Jun;8(6):LE20. 2002
Ferrari, G., Cusella-De, Angelis, G., Coletta, M., Paolucci, E., Sronaiuolo, A., Cossu, G., and Mavilio, F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, 1108-1110, 1998
Fraichard, A., Chassande, O., Bilbaut, G., Dehay, C., Savatier, P., and Samarut, J. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 108, 3181-3188. 1995
Gussoni, E., Soneoka, Y., Strickland, C.D., Buzney, E.A., Khan, M.K., Flint, A.F., Kunkel, L.M., and Mulligan, R.C. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401, 390-394. 1999
Herzenberg, L.A. and De Rosa, S.C. Monoclonal antibodies and the FACS: complementary tools for immunobiology and medicine. Immunol. Today. 21, 383-390, 2000
Jacson, K., Majka, S.M., Wang, H., Pocius, J., Hartley, C.J., Majesky, M.W., Entman, M.L., Michael, L.H., Hirschi, K.K., and Goodell, M.A. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cell. J. Clin. Invest. 107, 1-8, 2001
Julius, M.H., Masuda, T., and Herzenberg, L.A. Demonstration that antigen- binding cells are precursors of antibody-producing cells after purification with a fluorescence-activated cell sorter. Proc. Natl. Acdd. Sci. U.S.A. 69, 1934-1938, 1972
Kocher, A.A., Schuster, M.D., Szabolcs, M.J., Takuma, S., Burkhoff, D., Wang, J., Homma, S., Edwards, N.M., and Itescu, S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, remodeling and improves cardiac function. Nat. Med. 7, 430-436. 2001
Kramer, J., Hegert, C., Guan, K., Wobus, A.M., Muller, P.K., and Rohwedel, J. Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech. Dev. 92, 193-205. 2000
Kursad Turksen. Ottawa health research institute. Embryonic stem cells: methods and protocols. Humana Press Inc. 2002
Lagasse, E., Connors, H., Al Dhalimy, M., Reitsma, M., Dohse, M., Osborne, L., Wang, X., Finegold, M., Weissman, I.L., and Grompe, M. Nat. Med. 6, 1229-1234. 2000
Liu, S., Qu, Y., Stewart, T.J., Howard, M.J., Chakrabortty, S., Holekamp, T.F., and McDonald, J.W. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc. Natl. Acad. Sci. U. S. A. 97, 6126-6131. 2000
Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R., and McKay, R. Differentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similiar to Pancreatic Islets. Science. 292, 1389-1394. 2001
Maltsev, V.A., Rohwedel, J., Hescheler, J., and Wobus, A.M. Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types. Mech. Dev. 44, 41-50. 1993
Marwick C. Embryonic stem cell debate brings politics, ethics to the bench. J Natl Cancer Inst. Aug 15;93(16):1192-3. 2001
Mezey, E., Chandross, K.J., Harta, G., Maki, R.A., and McKercher, S.R. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 290, 1779-1782. 2000
Nakano, T., Kodama, H., and Honjo, T. In vitro development of primitive and definitive erythrocytes from different precursors. Science. 272, 722-724. 1996
Nishikawa, S.I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., and 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. 125, 1747-1757. 1998
Orlic,D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S.M., Li, B., Pickel, J., McKay, R., Nadal-Ginard, B., Bondine, D.M., Leri, A., and Anversa, P. Bone marrow cells regenerate infracted myocardium. Nature 410, 701-705. 2001
Petersen, B.E., Bowen, W.C., Patrene, K.D., Mars, W.M., Sullivan, A.K., Murase, N., Boggs, S.S., Greenberger, J.S., and Goff, J.P. Bone marrow as a potential source of hepatic oval cells. Science 284, 1168-1170. 1999
Potocnik, A.J., Nielsen, P.J., and Eichmann, K. In vitro generation of lymphoid precursors from embryonic stem cells. EMBO. J. 13, 5274-5283. 1994
Represa A, Shimazaki T, Simmonds M, Weiss S. EGF-responsive neural stem cells are a transient population in the developing mouse spinal cord. Eur J Neurosci. Aug;14(3):452-62. 2001
Risau, W., Sariola, H., Zerwes, H.G., Sasse, J., Ekblom, P., Kemler, R., and Doetschman, T. Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development. 102, 471-478. 1988
Robertson JA. Ethics and policy in embryonic stem cell research.Kennedy Inst Ethics J. Jun;9(2):109-36. 1999
Rohwedel, J., Maltsev, V., Bober, E., Arnold, H.H., Hescheler, J., and Wobus, A.M. Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev. Biol. 164, 87-101. 1994
Ruiz-Canela M. Embryonic stem cell research: the relevance of ethics in the progress of science. Med Sci Monit. May;8(5):SR21-6. 2002
Soria B. In-vitro differentiation of pancreatic beta-cells. Differentiation. Oct;68(4-5):205-19, 2001
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. Nov 10;95(23):13726-31, 1998
Strubing, C., Ahnert-Hilger, G., Shan, J., Wiedenmann, B., Hescheler, J., and Wobus, A.M. Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech. Dev. 53, 275-287, 1995
Theise, N.D., Nimmakayalu, M., Gardner, R., Illei, P.B., Morgan, G., Teperman, L., Henegariu, O., and Krause, D.S. Liver from bone marrow in humans. Hepatology 32, 11-16, 2000
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. Nov 6;282(5391):1145-7, 1998
Wiles, M.V. and Keller, G. Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development. 111, 259-267, 1991
Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., and Nishikawa, S.. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature. 408, 92-96, 2000