Original Articles Korean Circulation J 2002;32 8 : 제대혈에서유래한혈관내피전구세포의분화 조영애 1 백상홍 2 박효은 1 이영하 2 권희경 1 김영주 1 이수영 3 승기배 2 채장성 2 김재형 2 홍순조 2 최규보 2

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1 Original rticles Korean Circulation J 2002;328: 제대혈에서유래한혈관내피전구세포의분화 조영애 1 백상홍 2 박효은 1 이영하 2 권희경 1 김영주 1 이수영 3 승기배 2 채장성 2 김재형 2 홍순조 2 최규보 2 In vitro Differentiation of Endothelial Precursor Cells Derived from Umbilical Cord lood Young e Joe, PhD 1, Sang Hong aek, MD 2, Hyo-Eun Park, S 1, Young Ha Lee, MS 2, Hee-Kyung Kwon, S 1, Young-Ju Kim, S 1, Soo-Young Lee, PhD 3, Ki ae Seung, MD 2, Jang Seong Chae, MD 2, Jae Hyung Kim, MD 2, Soon Jo Hong, MD 2 and Kyu o Choi, MD 2 1 Cancer Research Institute, Catholic Research Institutes of Medical Sciences, 2 Division of Cardiovascular Medicine, Department of Internal Medicine, 3 Department of Chemistry, The Catholic University of Korea, College of Medicine, Seoul, Korea STRCT ackground and ObjectivesEx vivo expansion of endothelial cells is important when applying cell therapy to therapeutic angiogenesis in ischemic tissues. Endothelial precursor cells EPCs from the umbilical cord blood are one of adult stem cell. In order to establish the culture system for EPCs, we examined the effects of the media and matrix on the differentiation of a subset of mononuclear cells to endothelial cells, and analyzed their endothelial-lineage phenotype. Materials and MethodsMononuclear cells isolated from human umbilical cord blood were cultured in a chamber slide coated with fibronectin or gelatin in a M199 medium supplemented with 10 fetal bovine serum FS the normal medium or with 20 FS and ECGS the rich medium. Changes in the morphology and the attainment of DiI-ac-LDL uptake ability were examined during a 7 day period. The attached cells were immunostained for CD31, KDR, and vwf. ResultsThe fibronectin matrix gave rise to more attached cells than the gelatin matrix about 1.5 fold. The numbers of attached cells were no different between the normal medium and the rich medium at day 3 and 7, and were about 12 of the seeded mononuclear cells. However, the cell size and the numbers of longer spindle-shaped cells increased with the rich medium. Moreover, there was no increase in cellular population, but a 23 fold increase in the cellular size between day 3 and 7. bout 2040 of the attached cells acquired the DiI-ac-LDL uptake ability at day 3, whereas more than 85 of the attached cells could be stained with fluorescent DiI-ac-LDL at day 7 p The attached cells after being cultured for 7 days were stained moderately with the antibodies of CD31, or KDR. However, the cells at day 7 were only weakly immunostained with the vwf antibody, whereas more than 90 of cells were strongly stained at day 14. ConclusionThese results suggest that a subset of mononuclear cells derived from cord blood cells can give rise to cells with an endothelial cell-like phenotype, in vitro, at high percentages, which could be applied to in vivo vasculogenesis. Korean Circulation J 2002;328: KEY WORDSStem cellsendothelium, vascularcell differentitationvasculogenesis. 646

2 서론 in situ - - in vitro ex vivo 재료및방법 단핵구의분리 세포배양 647

3 - DiI-labeled acetylated-ldl(ac-ldl) 의섭취능조사 - 면역세포화학법 통계처리 결과 Matrix 성분이 EPC 배양에미치는효과 - 배지조성에따른 EPC의형상및세포성장의변화 Korean Circulation J 2002;328:

() or fibronectin () matrix for 7 days in

serum (FS normal medium -F) or with 20% FS

Moreover, there was no increase of cellular

4 Fig. 1. Comparison of matrix for the growth of endothelial precursor cell (EPC). Mononuclear cells were cultured on gelatin () or fibronectin () matrix for 7 days in M199 medium supplemented with 20% fetal bovine serum (FS) and endothelial cell growth supplement (ECGS). Fibronectin matrix gave rise to more attached cells than gelatin matrix. C D E F G H I J K L Fig. 2. Morphological changes and growth of endothelial precursor cell (EPC) in different medium. Mononuclear cells were cultured in M199 medium supplemented with 10% fetal bovine serum (FS normal medium -F) or with 20% FS and endothelial cell growth supplement (ECGS rich medium G-L) for 7 days. The rich medium elicited to increase of cell size and number of longer spindle-shaped cells. Moreover, there was no increase of cellular population but 2-3 fold increment of cellular size between day 3 and day 7. The images were photographed under the phase-contrast microscope during a period of 7 days serially. and G (day 1), and H (day 2), C and I (day 3), D and J (day 4), E and K (day 5), F and L (day 7) of culture. Magnification, 200. ar, 100 μm. 649

러 쌓이고, 내부에는 작은 구 모양의 단핵구들이 모여 성장하여, blood island 같은 구조도 관찰할 수 있었다 (Fig. 2). 세포 크기 방추형의 세포와 cobble stone 모양의 동그란 모양에 가까운 세포의 장축을 배양 3일째와 7일째 측정하였다. Table 1.

부착되어 성 20% FS+ECGS Spindle 065±17* Round 18±01 Spindle 075±06 ** Day 7 53±15 160±24 * 56±17 201±22** Cell shape round y/x<2 spindle y/x 2 (x short axis y long axis).

009, compared with normal medium, spindle cells, day 3, p<0.05, 20% FS+ECGS (rich medium), round cells at day 3, p<0.005, compared with rich medium, round cells at day 3, ** p<0.

FS fetal bovine serum, ECGS endothelial cell growth supplement 세포의 크기는 배지 조성에 관계없이 배양 3일

5 러 쌓이고, 내부에는 작은 구 모양의 단핵구들이 모여 성장하여, blood island 같은 구조도 관찰할 수 있었다 (Fig. 2). 세포 크기 방추형의 세포와 cobble stone 모양의 동그란 모양에 가까운 세포의 장축을 배양 3일째와 7일째 측정하였다. Table 1. Comparison of cell size of the attached cells between two different culture media 10% FS Round Day 3 16±01 7일에는 2 3배씩 증가되었다(Table 1). 부착되어 성 20% FS+ECGS Spindle 065±17* Round 18±01 Spindle 075±06 ** Day 7 53±15 160±24 * 56±17 201±22** Cell shape round y/x<2 spindle y/x 2 (x short axis y long axis). Cell size=maximal length minimal length/2. *, p<0.05, compared with 10% FS (normal medium), round cells at day 3, p<0.005, compared with normal medium, round cells at day 7, p<0.009, compared with normal medium, spindle cells, day 3, p<0.05, 20% FS+ECGS (rich medium), round cells at day 3, p<0.005, compared with rich medium, round cells at day 3, ** p<0.001, compared with rich medium, round cells at day 7, p<0.007, compared with rich medium, spindle cells at day 3, p<0.05, compared with normal medium, spindle cells at day 7. FS fetal bovine serum, ECGS endothelial cell growth supplement 세포의 크기는 배지 조성에 관계없이 배양 3일에 비하여 Fig. 3. Comparison of the number of the attached cells after cultured for 3 or 7 days in M199 medium supplemented with 10% FS or with 20% FS and ECGS. C D Fig. 4. Dil-ac-LDL uptake of the attached cells. Mononuclear cells were cultured on fibronectin matrix for 3 days ( and ) or 7 days (C and D) in M199 medium supplemented with 20% FS and ECGS. The attached cells were incubated with Dil-ac-LDL for 1 hour, washed with PS and photographed under phase-contrast microscope ( and C) and fluorescence microscope ( and D). Magnification, 200. ar, 100 μm. 650 Korean Circulation J 2002; 32(8):

장하는 세포중 영양 배지에서의 배양 7일째 방추형 세포 는 일반 배지의 세포보다 크다(201±22 vs.

세포 수 배양 3일째와 7일째 부착되어 있는 세포 수는, 세포 크기와는 달리 배지 조성에 관계없이 평균 200

이들 세포의 수는 seeding한 세포 수의 대략 12%에 해당하는 것이다.

Mononuclear cells were cultured on fibronectin matrix for up to

001, compared with rich medium at day 3.

배양시간에 따른 ac-ldl 섭취 능력의 변화 두 가지 배지 조성에서 7일간 배양후, 혈관 내피세포 표식자의

3 일째에는 부착되어 성장하는 세포들 중 20 40% 정도 가 ac-ldl을 섭취하고, 7일째에는 방추형의

FS Fig. 6. Immunocytochemistry of the attached cells.

6 장하는 세포중 영양 배지에서의 배양 7일째 방추형 세포 는 일반 배지의 세포보다 크다(201±22 vs. 160±24, p<0.05). 세포 수 배양 3일째와 7일째 부착되어 있는 세포 수는, 세포 크기와는 달리 배지 조성에 관계없이 평균 cells/mm2로 유사하였다(Fig. 3). 이들 세포의 수는 seeding한 세포 수의 대략 12%에 해당하는 것이다. 흥미로 운 것은 3일째와 7일째 모두 유사한 세포 수들이 부착 되어 자라는 것이 관찰되어, 3일과 7일 사이에는 세포 Fig. 5. Time-course change in ability of DiI-labeled acldl uptake. Mononuclear cells were cultured on fibronectin matrix for up to 8 days in M199 medium supplemented with 10% FS (normal medium) or with 20% FS and ECGS (rich medium). t each time point, the number of cells with Dil-ac-LDL uptake were counted. The results for ac-ldl uptakes are mean±sd from three indepedendent expreriments. * p<0.001, compared with normal medium at day 3. p<0.001, compared with rich medium at day 3. 증식보다는 세포 크기 및 형태의 변화가 관찰되었다. 배양시간에 따른 ac-ldl 섭취 능력의 변화 두 가지 배지 조성에서 7일간 배양후, 혈관 내피세포 표식자의 일종인 ac-ldl 섭취 능력을 조사하였다. 3 일째에는 부착되어 성장하는 세포들 중 20 40% 정도 가 ac-ldl을 섭취하고, 7일째에는 방추형의 세포들이 vwf C D E F G H I J K L CD31 KDR lgg HUVEC 20% FS+ECGS 10% FS Fig. 6. Immunocytochemistry of the attached cells. fter grown for 7 days in M199 medium supplemented with 10% FS (C, F, I and L) or with 20% FS and ECGS (, E, H and K), the attached cells were stained with a monoclonal antibody directed against human vwf (, and C), CD31 (D, E and F), KDR (G, H and I) or IgG (negative control J, K and L). Human umbilical endothelial cells (, D, G and J) were used as a positive control. Magnification, 200. FS fetal bovine serum, ECGS endothelial cell growth supplement. 651

7 Fig. 7. Immnunostaining for vwf dark brown reaction. The attached cells were stained stronger at day 14 compared with at day 7. Magnification, 200. 혈관내피세포계열성상의확인 고찰 Korean Circulation J 2002;328:

8 - in vitro 요약 배경및목적 : 653

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Can032.hwp

Can032.hwp Chromosomal Alterations in Hepatocellular Carcinoma Cell Lines Detected by Comparative Genomic Hybridization Sang Jin Park 1, Mahn Joon Ha, Ph.D. 1, Hugh Chul Kim, M.D. 2 and Hyon Ju Kim, M.D. 1 1 Laboratory