Journal of Veterinary Clinics (한국임상수의학회지)

The Korean Society of Veterinary Clinics (한국임상수의학회)
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Chondrogenesis of Mesenchymal Stem Cells Derived from Human Umbilical Cord Blood

사람 제대혈 유래 간엽줄기세포로부터 연골세포 분화

  • Koh, Phil-Ok (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Cho, Jae-Hyun (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Nho, Kyoung-Hwan (Laboratory of Stem cell and Tumor Biology, Department of Veterinary Public Health, and Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University) ;
  • Cha, Yun-Im (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Kim, Young-Ki (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Cho, Eun-Hae (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Lee, Hee-Chun (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Jung, Tae-Sung (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Yeon, Seong-Chan (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Kang, Kyung-Sun (Laboratory of Stem cell and Tumor Biology, Department of Veterinary Public Health, and Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University) ;
  • Lee, Hyo-Jong (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University)
  • 고필옥 (경상대학교 수의과대학 동물의학연구소) ;
  • 조재현 (경상대학교 수의과대학 동물의학연구소) ;
  • 노경환 (서울대학교 수의과대학 성체줄기세포연구센타) ;
  • 차윤임 (경상대학교 수의과대학 동물의학연구소) ;
  • 김영기 (경상대학교 수의과대학 동물의학연구소) ;
  • 조은혜 (경상대학교 수의과대학 동물의학연구소) ;
  • 이희천 (경상대학교 수의과대학 동물의학연구소) ;
  • 정태성 (경상대학교 수의과대학 동물의학연구소) ;
  • 연성찬 (경상대학교 수의과대학 동물의학연구소) ;
  • 강경선 (서울대학교 수의과대학 성체줄기세포연구센타) ;
  • 이효종 (경상대학교 수의과대학 동물의학연구소)
  • Published : 2009.12.31
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Abstract

In the current study, the mesenchymal stem cells (MSCs) isolated and propagated from the human umbilical cord blood (UCB) were tested for their capabilities of differentiation into chondrocytes in vitro. The mesenchymal progenitor cells (MPCs) collected from UCB were cultured in a low glucose DMEM medium with 10% FBS, L-glutamine and antibiotics. The human MSC colonies were positively stained by PAS reaction. When the immunophenotypes of surface antigens on the MSCs were analyzed by fluorescence-activated cell sorter (FACS) analysis, these cells expressed positively MSC-related antigens of CD 29, CD44, CD 90 and CD105, whereas they did not express antigens of CD14, CD31, CD34, CD45, CD133 and HLA-DR. Following induction these MSCs into chondrocytes in the chondrogenic differentiation medium for 3 weeks or more, the cells were stained positively with safranin O. We clearly confirmed that human MSCs were successfully differentiated into chondrocytes by RT-PCR and immunofluorescent stain of type-II collagen protein. These data also indicate that the isolation, proliferation and differentiation of the hUCB-derived MSCs in vitro can be used for elucidating the mechanisms involved in chondrogenesis. Moreover this differentiation technique can be applied to developing cell-based tissue regeneration or repair damaged tissues.

본 연구에서는 사람 제대혈로부터 간엽줄기세포를 분리하고, 이들을 체외에서 다량 증식시키며, 나아가 이들 간엽줄기세포를 특정한 세포로 분화시키고자 하였다. 사람의 제대혈로부터 단핵세포를 Ficoll-density gradient 법으로 분리하고, 이를 10% 우태아혈청, L-glutamnie, 및 항생제가 첨가된 DMEM 배양액과 Keratinocyte 배양액으로 $37^{\circ}C$ 5% $CO_2$ 배양조건에서 계대배양으로 증식시키고 현미경으로 줄기세포의 발달과 형태학적 성상을 확인하였으며, PAS 염색 및 PACS 분석으로 간엽줄기세포임을 확인하였다. 이들은 체외에서 연골세포로 분화를 유도하였고, 이들 분화된 줄기세포는 면역조직화학적 검사법으로 연골세포 특이 물질에 대한 Safranin O 염색법 및 Type II collagen 염색법을 실시하여 이들의 발현을 확인하였으며 RT-PCR을 실시하여 특이 mRNA 발현을 확인함으로서 연골세포로 분화된 것임을 확인하였다.

Keywords

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