Stem cell 은그기초연구뿐만아니라 transplantation 이나 regeneration 을통한 therapeutic medicine 으로주목받고있다. 이러한학문적, 산업적가치로인해 stem cell 에대한연구는치열한경쟁속에서진행되고있다. 특히, 1)stem cell 의 maintenance/self-renewal capacity 유지에대한 mechanism 과 2) 원하는특정 조직으로의 differentiation 을유도할수있는 method 개발등이주요연구 분야이다. Canonical Wnt signaling 은 beta-catenin 을통하여 target gene 으로알려진 cyclin D1, c-myc 등의발현을증가시킨다. 이러한 Wnt/beta-catenin signaling 은 stem cell 의 proliferation 과 cancer formation 에관여할뿐만아니라, 일부 stem cell 의 differentiation 과정에도관여한다고보고되고있다. 그리고, stem cell development 에서 Wnt5a 를비롯한 non-canonical Wnt signaling 의역할은거의알려진바가없다. 1. Human and mouse embryonic stem cell(esc) (Totipotent 하며, 모든 cell type 으로 differentiation 할수있는 unrestric stem cell) Paul Greengard 박사그룹은 human ESC 와 mouse ESC 의 gene profiling 을통해유전자발현패턴을조사한후 (25), '6-bromoindirubin-3'-oxime(BIO)' 라는 GSK3-beta inhibitor 를이용해서 activation 된 Wnt/beta-catenin signaling 이 human 과 mouse ESC 의 pluripotency 와 self-renewal capacity 를 유지시킨다는 결과를보였다 (2). 또한, 여러가지 APC mutant form 을이용해 얻어진결과는, 증가된 beta-catenin 의 intra-cellular dose 에의해 mouse ESC 의 three germ layer differentiation 이억제된다는것이다 (4). 위결과들에서보이는 Wnt signaling 의 effect 는 ESC 에서 differentiation 을억제하여 pluripotency 를
유지하고, self-renewal 즉 proliferation 을촉진시키는데관여하는것으로보인다. 이는비정상적인 Wnt signaling 의조절과관련된여러가지형태 Cancer 들의 induction 과 progression 을설명할수있는근거를제공하고있다. ( 예로 Wnt/beta-catenin signaling 은 intestinal epithelium 의 homeostasis 에필수적이며 (12), 지속적인 beta-catenin signaling activity 는 colon-carcinoma 의형성과관련이있다 (13).) 2. Tissue-specific stem cell (Multipotent 하며, adult tissue 의 homeostasis 와 repair 를담당하고, 특정조직으로만 differentiation 할수있는 restric' stem cell) 앞에서언급한것과같이 Wnt signaling 의 effect 는 embryonic stem cell 에서뿐만아니라, 각종 tissue-specific adult stem cell 에서도 proliferation 에관여한다. 그러나 cell lineage determination 과같은 differentiation 과정에도관여한다는보고들이있다. 이와같은결과는 mouse embryonic, neural, hematopoietic stem cells 의 gene information 을비교한결과 (26) 로보아, 서로다른유전자발현패턴으로인한것으로추측된다. 2-1 hematopoietic stem cell ( 대표적으로, Stanford Univ. 의 Roel Nusse 박사그룹 ) In vitro 에서 purification 한 Wnt3a protein 을처리하여 hematopoietic stem cell 의 self-renewal 에 Wnt/beta-catenin signaling 이관여한다는것 (19) 을보였다. 또한, 지속적인 activated beta-catenin 의발현과 inhibitor 인 Axin 과 mutant Fz9(CRD domain) 의발현을통하여 hematopoietic stem cell 에서 Wnt/beta-catenin
signaling 이 self-renewal capacity 를유지시킨다는 in vivo/vitro 결과들을보고하였다 (3). beta-catenin 을통하지않는 non-canonical Wnt signaling 의 stem cell 에대한연구는현재활발히진행중인것으로보인다. Murdoch, B. et al. (20) 에의하면, Wnt5a-conditioned media 를 human hematopoietic progenitor cell 을 engraft 한 mice 에 injection 한결과, reconstitution 이증가한것을관찰하였고, 이는 Wnt-5a signaling 이 hematopoietic stem cell expansion, engraftment 또는 survival 을촉진한다는것을보이고있다 2-2 intestinal stem cell ( 대표적으로, Hans Clevers 박사그룹 ) TCF-4-deficient mice 에서 fetal development 동안에 intestinal progenitor cell 이관찰되지않는결과들 (12,17) 을보였다. 더욱이, adult 에서 Wnt signaling 이 gut progenitor cell 에서 activation 된상태이고, Wnt signaling inhibitor 로알려진 Dickkopf-1(Dkk1) 를처리했을때, small intestine 과 colon 의 proliferation 이 억제되는결과들 (18) 로보아, Wnt signaling 은 intestinal stem cell 의 self-renewal 에관여한다고보여진다. 이러한결과들은 Wnt signaling activator 를이용하여 gut epithelium 의재생, 즉 homeostasis 를유지하고 (12), inflammatory bowel disease 같은질환에 adjuvant therapy 에이용될수있을것이다 (18). 2-3 skin stem cell
secreted Wnt inhibitor 인 DKK-1 을 skin specific K-14 promoter 를이용하여 epidermis basal cell 에서발현시킨 in vivo 실험결과, skin appendage 에속하는 hair follicle, teeth, mammary gland 의형성이억제된결과를얻었다 (23). Eaine Fuchs 박사그룹의연구결과는 active beta-catenin 의발현이 skin stem cell 의 population 의증가 multi potency 를유지시키고, TCf-3 과 Lef-1 와의 complex 를통하여 skin stem cell 의 lineage differentiation 에도관여한다는보고를하였다 (24). 2-5 muscle stem cell CD45+ muscle stem cell 을이용한 myogenesis 과정에서 Wnt signlaing 이 myogenic specification 을유도할수있으며, Wnt antagonist 인 sfrp2/3 을 regeneration muscle 에 injection 한결과 CD45+ stem cell 의 proliferation 과 myogenic specification 이감소한것을보였다 (21). 이러한연구는 muscle damage 즉 neuromuscular degeneratie disease 에대한 theraphy 에유용할거라여겨진다. 2-4 neural stem cell Wnt signaling 의 activation 이 embryonic stem cell 의 neuronal differentiation 을 억제하거나 (2,5), 발생과정시 neuronal precursor cell 의 expansion, 즉 proliferation 을촉진한다는연구결과들이보고들이있다 (6). 그러나 Wnt/betacatenin signaling 이 neuronal differentiation 에도관여한다는결과들도보고되고있다. Wnt signaling 의 activation 이 cortical neuron 의 differentiation 에관여한다는사실이 mouse in vivo 실험에서보고되었으며 (7), Wnt7a 가 neocortex 에서 neuronal precursor cell 의분화를유도하고 (8), Wnt/beta-catenin
signaling 이 embryonic stem cell 의 neuronal differentiation 에필요하다는결과가 보고되었다 (9). 2-5 neural crest stem cell Wnt signaling 은 neural crest stem cell 의 fate decision 에관여한다. Neural crest cell 은 peripheral nervous system(pns) 와 craniofacial, skin, heart structure 와관련된여러가지 cell type 을생성한다고알려져있다 (14), Activation 된 Wnt/beta-catenin signaling 은 early neural crest stem cell 을 sensory lineage 로 cell fate decision 을조절하지만, proliferation 에는영향을미치지않는다 (15,16).. pluripotency/ differentiation self-renewal multpotency (lineage determination) mouse embrionic stem cell o o human embrionic stem cell o o hematopoietic stem cell o o intestinal stem cell o o skin stem cell o o o muscle stem cell o o o neuronal stem cell o o o neural crest stem cell o
Reference 1. Kleber M. and Sommer L. (2004) Wnt signaling and the regulation of stem cell function. Curr. Opin. Cell Biol. 16: 681-687 2. Sato N., Meijer L., Skaltsounis L., Greengard P. and Brivanlou A. H. (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10: 55-63 3. Reya T., Duncan A. W., Ailles L., Domen J., Scherer D. C., Willert K. et al. (2003) A role for Wnt signalling in selfrenewal of haematopoietic stem cells. Nature 423: 409-414 4. Kielman MF, Rindapaa M, Gaspar C, van Poppel N, Breukel C, van Leeuwen S, Taketo MM, Roberts S, Smits R, Fodde R: Apc modulates embryonic stem-cell differentiation by controlling the dosage of b-catenin signaling. Nat Genet 2002, 32:594-605. 5. Haegele L., Ingold B., Naumann H., Tabatabai G., Ledermann B. and Brandner S. (2003) Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression. Mol. Cell. Neurosci. 24:
696-708 6. Chenn A. and Walsh C. A. (2002) Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297: 365-369 7. Maretto S., Cordenonsi M., Dupont S., Braghetta P., Broccoli V., Hassan A. B. et al. (2003) Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc. Natl. Acad. Sci. USA 100: 3299-3304 8. Hirabayashi Y., Itoh Y., Tabata H., Nakajima K., Akiyama T., Masuyama N. et al. (2004) The Wnt/beta-catenin pathway 8 F. Ille and L. Sommer Wnt signaling directs neuronal differentiation of cortical neural precursor cells. Development 131: 2791-2801 9. Otero J. J., Fu W., Kan L., Cuadra A. E. and Kessler J. A. (2004) Beta-catenin signaling is required for neural differentiation of embryonic stem cells. Development 131: 3545-3557 10. Pinto D, Gregorieff A, Begthel H, Clevers H: Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev 2003, 17:1709-1713.
11. Alonso L, Fuchs E: Stem cells in the skin: waste not, Wnt not. Genes Dev 2003, 17:1189-1200. 12.van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Batlle E, Coudreuse D, Haramis AP et al.: The b-catenin/tcf-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 2002, 111:241-250. 13. Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, Oshima M,Taketo MM: Intestinal polyposis in mice with a dominant stable mutation of the b-catenin gene. EMBO J 1999, 18:5931-5942. 14. Le Douarin N. M. and Dupin E. (2003) Multipotentiality of the neural crest. Curr. Opin. Genet. Dev. 13: 529-536 15 Hari L., Brault V., Kleber M., Lee H. Y., Ille F., Leimeroth R.et al. (2002) Lineage-specific requirements of beta-catenin in neural crest development. J. Cell Biol. 159: 867-880 98 16. Lee H. Y., Kleber M., Hari L., Brault V., Suter U., Taketo M. M. et al. (2004) Instructive role of Wnt/beta-catenin in sensory fate specification in neural crest stem cells. Science 303: 1020-1023
17. Korinek, V. et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nature Gene t. 19, 379-383 (1998). 18. Kuhnert, F. et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc. Natl Acad. Sci. USA 101, 266-271 (2004). 19. Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448-452 (2003). Purification of WNTs and demonstration of their potential effects on stem cells. 20 Murdoch, B. et al. Wnt-5A augments repopulating capacity and primitive hematopoietic development of human blood stem cells in vivo. Proc. Natl Acad. Sci. USA 100 21. Polesskaya, A., Seale, P. & Rudnicki, M. A. Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration. Cell113, 841 852 (2003). 22.Liu, B. Y., McDermott, S. P., Khwaja, S. S. & Alexander, C. M. The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells. Proc. Natl Acad. Sci. USA101, 4158 4163 (2004).
23. Andl, T., Reddy, S.T., Gaddapara, T., and Millar, S.E. 2002. WNT signals are required for the initiation of hair follicle development. Dev.Cell 2: 643.653. 24. Merrill, B.J., Gat, U., DasGupta, R., and Fuchs, E. 2001. Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin. Genes & Dev. 15: 1688.1705. 25.Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev. Biol.260, 404 413 (2003). 26. Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C. & Melton, D.A. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science298, 597 600 (2002). 27. Kleber M, Sommer L. Wnt signaling and the regulation of stem cell function. Curr Opin Cell Biol. 2004 Dec;16(6):681-7. 28 Brivanlou AH, Gage FH, Jaenisch R, Jessell T, Melton D, Rossant J Stem cells. Setting standards for human embryonic stem cells. Science. 2003 May 9;300(5621):913-6.
29. Ille F, Sommer L. Wnt signaling: multiple functions in neural development. Cell Mol Life Sci. 2005 Feb 28; 30 Beachy PA, Karhadkar SS, Berman DM Tissue repair and stem cell renewal in carcinogenesis. Nature. 2004 Nov 18;432(7015):324-31. 31. Ding S, Schultz PG. A role for chemistry in stem cell biology. Nat Biotechnol. 2004 Jul;22(7):833-40. 32. Alonso L, Fuchs Stem cells of the skin epithelium.proc Natl Acad Sci U S A. 2003 Sep 30;100 Suppl 1:11830-5.