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1 Biomaterials Research (2006) 10(3) : Biomaterials Research 7 The Korean Society for Biomaterials x y» s w Recent Advances in Umbilical Cord Blood-derived Multipotent Stem Cell Research y wá yáw z* Soo Han Hwang, Il Ho Yang, and Hoon Han* Â Š Research Institute of Biotechnology, Histostem Co., Seoul , Korea (Received November 28, 2005/Accepted July 14, 2006) Human umbilical cord bloodg (UCB)-derived multipotent stem cells are regarded as valuable sources for cell transplantation and cell therapy. These cells, under appropriate culture conditions, can differentiate into a variety of mesenchymal lineage cells such as osteoblasts, chondrocytes, adipocytes, skeletal myoblasts and neuronal cells, and these differential gene expression profiles can be analyzed by DNA microarray. Based on their large ex vivo expansion capacity as well as on their differentiation potential, clinical transplantation of UCB- derived multipotent stem cells may become a suitable source for tissue engineering and regenerative medicine. Key words: Umbilical cord blood, Multipotent stem cells, Mesenchymal stem cells f Š il hf igš j Š f g Š ff, Œ ƒh f l Œ ff } Œ f l f. ~ il Œ j ff f Š f il Œ f f l, u h (~j) igš h Œ (Umbilical Cord Blood) Œ f f j ig Š f lf, h Œf j f d ef f f (Figure 1). h Œf, g f f h f, f h Œ hœ, Œ, Œ Œ Š iœ j (Hematopoietic stem cell) ftf l g Œ f Œ j (Multipotent stem cell) Š e f, Š l il 1-6) f g ~ h Œf fdš f (j j (Mesenchymal Stem Cell) f f f h f f). Šl, j f ef f j (Embryonic Stem Cell) e hf Š h f Œ f ŠŠ i f f f h f x d j Š Š Š hf d h. Š, e hf h, l f t j ef eœšl f f f l x *sf hf: hhanscord@seoulcord.co.kr Š d h f, ƒ f tj h f Œ f l ~f d Š ff e Š dhf d d hf., Œ f Œ f e h Œ j g fš(regenerative Medicine)f hf Œd f f ff~ fšf Š hf v f. x» s p x x s 1960, Wiskott-Aldrich l f f Š f, f f Œ f Œ lœ g 7) Œ lœf x eš x f d. f f eš g f hf f 8) HLA (Human Leukocyte Antigen : ilhš Še) fxš Œh l, hšf fš hhš Œh ff hš f, HLA fš Œ ff j d, f e d HLA hf fxšl ff dš x Š Š t, h Œ f iœ igš f h, 1988 ut 9) Fanconi Œ Œff 5 f h Œ f 97

98 Œ ŠÁ fœáš Figure 1. Embryonic stem cell and adult stem cell. Š f, l l hf 8,000 f f h Œ 10) f f Š. 1996 e jl g Œ Œf f h Œ iœ f f sf, 1998 ut h Œ f f r f, l l 250 f f h Œ f f Š.

2 98 Œ ŠÁ fœáš Figure 1. Embryonic stem cell and adult stem cell. Š f, l l hf 8,000 f f h Œ 10) f f Š e jl g Œ Œf f h Œ iœ f f sf, 1998 ut h Œ f f r f, l l 250 f f h Œ f f Š. 11) k x y» s e Œ,, s Šl f Œ lœ Š h Œ iœ f f f l f 2000 t h Œ Œ j f Š tl. l fš ut 2003 Œf Œf Š ~f h Œ j x f, 2004 s Šl Œf,,, Œf Š h Œ Œ j x l fš f x l x h Š f hf l f Œhf r f, x x x f d f. x y» s xk h Œ j swš l dš f f, hf Š g Š f. Š, j f f iš HLAehf fš j 2-3 units Š dš Š. h Œ Š Œ j f Œ~ f f, 2-3r f ~ o f iš e Œ~ Š f (Figure 2A). Š, 14%f f Š, l f G 0 /G 1 j f v (Figure 2B), 60 zf 2 l f, h Œ j f l f j d e Š f (Figure 2C). x y» s txx r ~ h Œ j f ~ d f ƒf Šef FACS Š, f f CD13, CD29 (β1-integrin), CD31, CD44, CD45, CD49e (α5 integrin), CD54 (ICAM-1), CD90 (Thy-1), ASMA (alpha smooth muscle actin), CD105/SH2/endoglin, CD73/SH3 HLA class I Še fl, CD14, CD34 f iœ f ƒ f Še CD49d (α4 integrin), CD106 (VCAM-1), HLA- DR Šef f f, iœ h f (Figure 3). f f h Œ igš Œ j 12) Š Šl Š f l f h Œ Œ j igšl Š l, f j f f hf h Œf fdš j f Š, hhšl Š i Š h Œ j Š f. x y» s y h Œ j Œ e l 2jf ~, f gf f x, alkaline phosphatase (ALP) Œ Œ, Alizarin Red S ŒŠ f Š xr x f l f Œf (Figure Biomaterials Research 2006

제대혈 다분화능 줄기세포 Morphology and proliferative characteristics of adherent cells isolated from UCB. (A) Morphology of a homogeneous cell population in the fourth passage that was isolated from human UCB.

3 제대혈 다분화능 줄기세포 Morphology and proliferative characteristics of adherent cells isolated from UCB. (A) Morphology of a homogeneous cell population in the fourth passage that was isolated from human UCB. Magnification 40x. (B) Cell cycle analysis by flow cytometry. Percentage of cells in G0/G1, S, and G2/M phases were indicated. (C) Growth curve of UCB-derived cells. The cell numbers of three different cell cultures (n=3) were measured by a trypan blue exclusion assay and expressed as means ± SD of three experiments. An average population doubling time was estimated to be around 30 h. 99 Figure 2. 4A). 또한, 골세포분화 유도배지를 첨가한 다음부터 골모세포 에 특이한 osteopontin, ALP 유전자 발현이 조금씩 증가되면 서 세포외기질을 만드는 골모세포로 분화된다(Figure 4B). 제대 혈 다분화능 줄기세포를 연골세포분화 유도배지에서 5주일동안 배양시키면 다층의 기질세포 모습이 보이며, Safranin Red O 염색을 통해 sulfated proteoglycan 축적이 확인되며(Figure 4C), 연골세포의 특이한 전사인자인 SOX9, Runx2, aggrecan 유전자 발현이 증가된다(Figure 4D). 제대혈 다분화능 줄기세 포를 지방세포분화 유도배지에서 2주일동안 배양시키면 세포의 핵 주위에 지방 입자가 형성되어 Oil Red O 염색에 의해 확 Immunophenotypes of adherent cells derived from UCB. Flow cytometric analysis of hematopoietic (CD14, CD34, and CD45), endothelial (CD31 and CD106), and mesenchymal (CD44, CD49e, CD54, CD90, and ASMA) lineage markers as well as human leukocyte antigens(hla-abc and HLA-DR) on UCB-derived adherent cells. An open profile represents an isotype control for background fluorescence and a shaded one shows a positive signal. Figure 3. 인되며(Figure 4E), 지방세포의 특이한 전사인자인 PPARγ 유전 자 발현이 증가된다(Figure 4F). 제대혈 다분화능 줄기세포를 골격근육세포 분화 유도배지에서 배양시키면 Myosin Heavy Chain (MyHC)의 발현이 3주일 후부터 나타나기 시작하고 6 주일이 경과할 때까지 지속적으로 증가됨으로써 세포들의 절반 이상이 MyHC를 발현하며(Figure 4G), 골격근육세포의 특이한 전사인자인 MyoD, myogenin 유전자 발현이 1주일 이내에 증가된다. 또한, 이 유전자들의 발현이 2주일 후부터 급격히 감소하기 시작하는 것은 MyoD와 myogenin 유전자가 골격근 Vol. 10, No. 3

4 황수한 양일호 한 훈 100 육세포의 초기 발생과정에 관여한다는 사실과 일치한다(Figure 4H). 제대혈 다분화능 줄기세포를 간세포 분화 유도배지에서 1-2주일 배양시키면Albumin, AFP, CK-18, GS 유전자들의 발 현이 시작하고, 분화 과정이 성숙단계에 이르면 HGF, PEPCK, c-met, TAT, CPS 유전자들이 발현되는데(Figure 5A), 세포배양 을 시작한 다음 1-2주일이 경과되었을 때 WB와 IF 방법으로 단백질 발현량을 분석하면 미분화세포들은 Albumin, AFP, CK18, CK-19 모두 음성이지만, 2주일동안의 세포 초기 분화 과 정과 2주일 동안의 세포 성숙 분화 과정을 모두 수행시키면 Albumin, AFP, CK-18, CK-19 모두 양성이어서, 제대혈 줄기 세포는 배아 줄기세포와 마찬가지로 다분화능력을 가진 것으로 판단된다(Figure 5B). 제대혈 다분화능 줄기세포의 DNA Microarray 자료분석 사람의 세포는 약 30,000개 정도의 동일한 유전자를 가지고 있지만, 각각의 세포에서 모든 유전자들이 동시에 발현되는 것 은 아니며, 필요에 따라서 어떤 유전자가 발현되는 가를 연구 하는 것이 매우 중요하다. 유전자 발현 분석을 위해서 northern blot, RT-PCR 방법 등이 많이 사용되었지만, 최근에 는 짧은 시간 내 다량의 유전자 발현 연구를 시행할 수 있는 serial analysis of gene expression (SAGE) 기술이 발달해 제 대혈 줄기세포의 유전자 발현 양상을 짧은 시간내에 많이 밝 혀냈고, 유전자를 고밀도로 부착시킨 DNA microarray 실험을 통해, 제대혈 CD34 조혈모줄기세포에서stem cell receptor (c-kit)를 포함한 139개 특이 유전자 발현이 확인되었다. 또 한, 제대혈 CD34 다분화능줄기세포에서는 47개 특이 유전자 가 분화된 세포보다 50배 이상 발현되는 것이 확인되었는데, 이 중에서 28개(>65%) 유전자들은 extracellular matrix (ECM), cytokine, cytoskeleton-associated protein들과 연관된 단백질을 형성하는 유전자들이었기 때문에, 줄기세포의 기능 형 성에 가장 중요한 것으로 판단되었다. 또한, 제대혈 줄기세포 의 유전자 발현을 정상화시켰을 때, average normalized intensity가 가장 높은 상위 50개 유전자 중에서 24개는 서로 다른 ribosomal protein인 것으로 확인되었다. 이 결과를 통해 서, 제대혈 줄기세포는 배아 줄기세포와 비슷한 양식의 다분화 능을 가지고 있다는 것이 증명되었고, 사람의 줄기세포 유전자 발현에 관한 분자 수준의 기초 연구가 큰 도움을 받을 수 있 게 되었다. 또한, 제대혈에 포함된 줄기세포의 세포수가 부 족한 문제를 근본적으로 해결하기 위하여 제대혈 줄기세포를 체외에서 증식시킨 후 대량의 세포를 이식하는 방법들이 세계 적으로 활발히 연구되고 있으며, 여러가지 성장인자들을 이용 한 배양기술과 3차원적인 배양지지체를 사용한 배양기술을 비 롯하여 feeder layer없이 배양하는 방법, 동물의 혈청을 사용하 지 않고 배양하는 방법 등에 관한 연구가 진행되고 있으나, 실 용화까지는 미치지 못하고 있는 실정이다. 특히, 희귀한 HLA 를 갖는 불치, 난치병 환자를 치료하려면 냉동보관된 특정 제 대혈을 해동, 증폭시켜 일부는 치료에 사용하고 일부는 다시 동결 보관하여야 하므로 이 분야의 연구는 필수적이라 하겠다. 12) 13) + 14) Mesengenic differentiation of UCB-derived hmscs. (A) Cytochemical analysis of osteogenic differentiation. For detection of ALP activity, the cells incubated for 2 weeks in regular culture medium(a, control) and osteogenic medium(b) were investigated by ALP staining. For visualizing calcium deposits, the cells cultured for 2 weeks in culture medium(c, control) and osteogenic medium (d) were stained by alizarin red S. Magnification 40x. (B) RT-PCR analysis of gene expression of ALP and osteopontin in the cells incubated in osteogenic medium during the first one week. (C) Histochemical analysis of chondrogenic differentiation in pellet culture. The pelleted cells were incubated for 5 weeks in regular culture medium (a, control) and chondrogenic medium (b), and stained with safranin red O to visualize sulfated proteoglycan. Magnification 40x. (D) RT-PCR analysis of gene expression of SOX9, Runx2, and aggregan in the cells incubated in chondrogenic medium during four weeks. (E) Phase contrast microscopic images of the cells cultured for 2 weeks in adipogenic medium (a) and their positive staining with oil-red O (b). Magnification 100x. (F) RT-PCR analysis of gene expression of PPARγ. (G) Phase contrast microscopic image (a) and flow cytometric analysis of expression of MyHC in cells incubated for 6 weeks in regular culture medium (b, control) or myogenic medium (c). Magnification 100x. (H) RT-PCR analysis of MyoD and myogenin in cells incubated for 2 weeks in myogenic medium. Figure 4. Biomaterials Research )

h Œ Œ j 101 Figure 5. RT-PCR and WB analyses of the temporal expression pattern of hepatocyte-specific markers during hepatic differentiation of UCB-derived cells.

5 h Œ Œ j 101 Figure 5. RT-PCR and WB analyses of the temporal expression pattern of hepatocyte-specific markers during hepatic differentiation of UCB-derived cells. (A) RT-PCR analysis showed that UCB-derived cells could express a number of hepatocyte-specific genes in a timedependent manner. Among them, albumin, AFP, CK-18, and GS are known as early hepatogenic markers while TAT is known to be a late marker in hepatogenesis. It is worthy to note that regardless of the time point of the first appearance, mrna expression for all tested genes appeared to be significantly promoted in maturation medium containing OSM. (B) WB analysis indicated that these hepatocytespecific markers were actively synthesized during hepatic differentiation. x» s w y x» s e ƒ h Œf h f ff Œ Š h f ff e ~ j x g hšš ef g ff, f f h Œ j ŒŠŠ Š f Œf f Š f h Œ j f hd e h Œ i Œ f Œf l Œ f., Œf dš v Š f j, d x Š f fš iš h Œ j dš f x eš hd l Š f, eh f x eš j td l ~ llt fdš rehf f hd ~ f f. hf, h Œ j iœ,,, l, f Œ Œ f l, h Œ j f z Š Œf Šd Š f ~ l Œf f ~ j fšf f Œ f, x Œff x hdš d tf Ž ilf t f f l ff, h Œ Œ j x d f f f h j d Š (Table 1) ) n ƒ w x» s s f Š f Œf h Œ j j Š f, f 1jf l f, 3jf o s v do sv l d f g f dlf f, f 17), j f ff f h lf, Œ f Œ fš Œ f v i h Œh fš Œ f h (Buerger s Disease) Œf h Œ j j Š f, tœ f Œ hf d v Œ e f lf l 1 f g. 18) Š, f l f f Šl Œ (Liver Cirrhosis) Œf h Œ j j Š f, 2jf, Œ, l f l Œ l Table 1. Umbilical cord blood as multipotent stem cells for allogeneic transplantation 1. h Œf f j ef, h Š l. 2. h hf w Š, e hf hhf h l. 3. f f d h. 4. h Œf hhš Œf h Š f. 5. h Œf f f, l f Œ. 6. h Œ f f f hf f f j ff e Š. ^U G G v SG G sg G ƒœg y G n U _U G G ƒ G n G ŒSG Œm mg jg j U 9. g f ŠdŠ, fš h Œf fdf Š. Vol. 10, No. 3

6 102 Œ ŠÁ fœáš f f i i l f r, d f } 30% l. f } dh f f l ft d h h e dh l f l Œf h Œ j f f, (BMD) 5.6% l f, f x f 63% rlš Š lœf wšf (Alzheimer s Disease) Œf h Œ j j Š l f Œ f Œf. f f, Œf x hdš h Œ j if f l, f f Š lœf x Š f Š f (Figure 6). ev w Œl(Amyotrophic Lateral Sclerosis, ALS)f fif f s f d sf,, t f f ef ev fš, 3-5 f Œ f Š Š f, 2000 f Š h Œ x Š f h, 19) Œf h Œ j j f, x f f fš e f l Œ igx f Œ Š f, f Œ f ƒh ef Œ Œ g ff f f f f h (Cerebral infarction) Œf h Œ j j f, Œff 27% Motor Grade f 1 f } Š Œh ~, Œff 37% f 1 fšf f Œh ~ f f. Š, f Šf fdg fš f f dš Œ x ihš (Diabetes mellitus) Œf h Œ j j f, f f Šd f 20% } Œ f h Œ, l l g Š f fi Œf f f x f, Œf h Œ j j Š f, Œff 65-70% f f x f l f Œf. Š, v Œ Š hf f f f ~l Š lœf f l (Autism) gf f f } hš Figure 6. Clinical applications of umbilical cord blood-derived multipotent stem cells. f l ff f g f f h Œf, f f t f Œ f Š Šl, Œ e,, g, e, h f Šil z lf l i lf g f vh Š el l Œf, Š ef lœf f Š 6 e f l h lœ l t Š l (Chronic Fatigue Syndrome)Œf, Œl, Œf h Œ j x. x» s ftf g ilf f h ŠŠl Š d g, ilf j j Š f z x Š g fšf fšf Š f f g fšf Š Š hš e h f j f ef h Œff f l ff hf ŒdŠ f f f i Š. t m, h Œ j vvš ef Š ; h Œf hš Š j ef Š t j Š f f h Œ j f vvf e f, f x ŠdŠ f f h Œ j vv ef Š. m, h Œ fšf jf jdš f ; Š HLAehfŒf h Œf Œ Š f Š HLAŒf l Œf Š ehf hf back-upf Š f. m, h Œ f f v Š. l, Œ Š, f Š h Œf ŒdŠ j f f hf Œ h Œf f hdf d Œ z hf gf j hf r vš f f ff f. l, if x Š f s elœ, s lœ, ij, vœ, ~, wšf lœ, s f lœf eh x, h l, Œ l, f f ŠdŠ lœ f hf eff f f lœf x h f w f f fdš gf s Šh jf Š f. g fšf g h Š 1 f Š f }21 f jd f Œ f,, f f Š g f hf re h Œ j f jd f iš f. 21 f fš f fef h Œ f g f Œ ~ h f Š hsf f d leš, f ~f Š rfhf hhf h Š Š, f t f ŒŠ Š f hhš Œ th vš Š. Biomaterials Research 2006

7 h Œ Œ j 103 h Œ Œ j vvš j ehf f x hd f f Œf, f f i Š h Œ Œ j dš f l d f. tm, x x f x ~ f ilf g f f d hf ŠŠ eš h Œ j x Š. m, hhf d f h Œ j f x Š. m, h Œ j x g, f Œ g f f tš g f f Š l d f u Š v f. m, lœf t h Œ j x ŠŠ f ehf. fe ˆ g ( )f le fš f f. š x 1. W. A. Noort, A. B. Kruisselbrink, P. S. in t Anker, M. Kruger, R. L. van Bezooijen, R. A. de Paus, M. H. Heemskerk, C. W. Lowik, J. H. Falkenburg, R. Willemze, and W. E. Fibbe, Mesenchymal stem cells promote engraftment of human umbilical cord bloodderived CD34(+) cells in NOD/SCID mice, Exp. Hematol., 30, (2002). 2. P. S. in t Anker, W. A. Noort, A. B. Kruisselbrink, S. A. Scherjon, W. Beekhuizen, R. Willemze, H. H. Kanhai, and W. E. Fibbe, Nonexpanded primary lung and bone marrow-derived mesenchymal cells promote the engraftment of umbilical cord blood-derived CD34(+) cells in NOD/SCID mice, Exp. Hemato., 31, (2003). 3. M. Angelopoulou, E. Novelli, J. E. Grove, H. M. Rinder, C. Civin, L. Cheng, and D. S. Krause, Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice, Exp. Hematol., 31, (2003). 4. O. N. Koc, S. L. Gerson, B. W. Cooper, S. M. Dyhouse, S. E. Haynesworth, A. I. Caplan, and H. M. Lazarus, Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy, J. Clin. Oncol., 18, (2000). 5. M. F. Pittenger, A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simonetti, S. Craig, and D. R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science, 284, (1999). 6. J. J. Minguell, A. Erices, and P. Conget, Mesenchymal stem cells, Exp. Bio. Med., 226, 44, (2001) 7. F. H. Bach, R. J. Albertini, P. Joo, J. L. Anderson, and M. M. Bortin, Bone marrow transplantation in a patient with the Wiskott-Aldrich syndrome, Lancet, 292, (1968). 8. M. M. Bortin, M. M. Horowitz, and A. A. Rimm, Increasing utilization of allogeneic bone marrow transplantation, Ann. Int. Med., 116, (1992). 9. H. E. Broxmeyer, G. W. Douglas, G. Hangoc, S. Cooper, J. Bard, D. English, M. Arny, L. Thomas, and E. A. Boyse, Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells, Proc. Natl. Acad. Sci. USA., 86, (1989). 10. E. Gluckman, H. E. Broxymeyer, A. D. Auerbach, H. S. Friedman, G. W. Douglas, A. Devergie, H. Esperou, D. Thierry, G. Socie, P. Lehn, S. Cooper, D. English, J. Kurtzberg, J. Bard, and E. A. Boyse, Hematopoietic reconstitution in a patient with Fanconi s anemia by means of umbilical cord blood from an HLA-identical sibling, N. Engl. J. Med., 321, (1989). 11. Œ, h Œ iœ f -, g, Š ŒŠ l, 15, (2004). 12. E. J. Gang, S. H. Hong, J. A. Jeong, S. H. Hwang, S. W. Kim, I. H. Yang, C. Ahn, H. Han, and H. Kim, In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells, Biochem. Biophys. Res. Commun., 321, (2004). 13. S. H. Hong, E. J. Gang, J. A. Jeong, C. Ahn, S. H. Hwang, I. H. Yang, H. K. Park, H. Han, and H. Kim, In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells, Biochem. Biophys. Res. Commun., 330, (2005). 14. X. He, V. Gonzalez, A. Tsang, J. Thompson, T. Tsang, and D. T. Harris, Differential gene expression profiling of CD34+ CD133+ umbilical cord blood hematopoietic stem progenitor cells, Stem Cells And Development, 14, (2005). 15. J. A. Jeong, S. H. Hong, E. J. Gang, C. Ahn, S. H. Hwang, I. H. Yang, H. Han, and H. Kim, Differential gene expression profiling of human umbilical cord blood-derived mesenchymal stem cells by DNA microarray, Stem Cells, 23, (2005). 16., j x eš h Œf fd, Šf Œ l, 47, (2004). 17. K. S. Kang, S. W. Kim, Y. H. Oh, J. W. Yu, K. Y. Kim, H. K. Park, C. H. Song, and H. Han, A 37-year-old spinal cord-injured female patient, transplanted of multipotent stem cells from human UC blood, with improved sensory perception and mobility, both functionally and morphologically : a case study, Cytotherapy, 7, (2005). 18. S. W. Kim, H. Han, G. T. Chae, S. H. Lee, S. Bo, J. H. Yoo, Y. S. Lee, K. S. Lee, H. K. Park, and K. S. Kang, Successful stem cell therapy using umbilical cord blood-derived multi-potent stem cells for Buerger s disease and ischemic limb disease animal model, Stem Cells, in press. 19. N. Ende, F. Weinstein, R. Chen, and M. Ende, Human umbilical cord blood effect on sod mice(amyotrophic lateral sclerosis), Life Science, 67, (2000). Vol. 10, No. 3

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Biomaterials Research (2006) 10(4) : 187-195 Biomaterials Research 7 The Korean Society for Biomaterials DNA Chip w The Market Analysis of DNA Chip 1Á 1 Á 1 Á 1 *Á 2 * Jong-Ku Son, Young Seo Park,