대한수혈학회지 (The Korean Journal of Blood Transfusion)

  • 제21권3호
  • /
  • Pages.280-288
  • /
  • 2010
  • /
  • 1226-9336(pISSN)
  • /
  • 2383-6881(eISSN)
대한수혈학회 (The Korean Society of Blood Transfusion)
권호 표지

골수처리키트 내 잔존하는 골수세포로부터 중간엽줄기세포의 분리

Isolation of Mesenchymal Stem Cells from the Mononuclear Cells Remaining in the Bone Marrow Processing Kit

  • 김신영 (연세대학교 의과대학 진단검사의학교실) ;
  • 허준석 (연세대학교 의과대학 세포치료센터) ;
  • 김한수 (연세대학교 의과대학 진단검사의학교실) ;
  • 김현옥 (연세대학교 의과대학 진단검사의학교실)
  • Kim, Sin-Young (Department of Laboratory Medicine, Yonsei University College of Medicine) ;
  • Heo, June-Seok (Yonsei Cell Therapy Center, Yonsei University College of Medicine) ;
  • Kim, Han-Soo (Department of Laboratory Medicine, Yonsei University College of Medicine) ;
  • Kim, Hyun-Ok (Department of Laboratory Medicine, Yonsei University College of Medicine)
  • 투고 : 2010.11.18
  • 심사 : 2010.12.12
  • 발행 : 2010.12.31
⟨ 이전 논문 다음 논문 ⟩

초록

배경: 중간엽줄기세포는 지속적인 자기 재생산(self renewal) 능력과 함께 다양한 조직으로 분화할 수 있는 유연성(plasticity)을 가지고 있어 재생의학, 조직공학 및 유전자 치료의 매력적인 연구소재로 주목을 받으며 많은 연구가 진행되고 있다. 골수에서 중간엽줄기세포를 분리하는 경우 다른 조직에 비해 일정 수준 이상의 질을 가진 충분한 양을 회수할 수 있다. 그러나 골수를 건강한 지원자로부터 단순히 연구목적으로 확보하기는 현실적으로 매우 어렵다. 따라서 본 연구에서는 폐기되는 골수처리키트에 남아있는 골수로부터 연구목적용 골수유래 중간엽줄기세포를 분리하고자 하였다. 방법: 건강한 골수공여자로부터 채집된 골수를 COBE 2991 Cell Processor (CaridianBCT Inc.)를 이용하여 적혈구와 혈장의 분리제거를 시행한 후, 골수처리키트에 남아 있는 골수를 채집 2시 간 내에 회수하였다. 회수된 골수는 밀도구배원심분리를 이용하여 백혈구연층을 분리하고 부착세포의 증식이 일어날 수 있도록 배양하였다. 부착세포의 세포충실도가 70~80%가 되는 경우 회수하여 계대배양하거나 액체질소에 냉동보관하였다. 분리 배양한 중간엽줄기세포의 성상을 파악하기 위해 유세포분석 및 골세포, 지방세포, 연골세포로의 분화 여부를 확인하였다. 결과: 골수처리키트 내에 남아있는 골수로부터 회수한 단핵세포의 수는 평균 $1{\times}10^8$개였으며 모두에서 중간엽줄기세포를 분리 배양할 수 있었다(n=4). 또한 1회의 계대배양을 통하여 약 3주 후 평균 $3{\times}10^7$개의 중간엽줄기세포를 얻을 수 있었다. 유세포분석 상 중간엽줄기세포의 표지자인 CD29, CD44, CD73, CD90, CD105는 모두 강하게 발현하였으며, 조혈모세포의 표지자인 CD34와 CD45, 내피세포의 표지자인 CD31과 단핵구의 표지자인 CD14은 발현되지 않았다. 중간엽줄기세포의 기능적 측면을 평가하기 위하여 골세포, 지방세포 및 연골세포로의 분화를 유도하였으며, 세 가지 세포로 모두 분화가 가능함을 확인하였다. 결론: 골수이식 시 폐기되는 골수처리키트로부터 중간엽줄기세포를 다량 확보할 수 있었으며, 추후 중요한 중간엽줄기세포의 공급원으로서의 가능성을 확인하였다.

Background: Mesenchymal stem cells (MSCs) that are capable of extensive self renewal and differentiation have attracted great attention as a promising tool for regenerative medicine and tissue engineering. Although MSCs can be isolated from various tissues, bone marrow currently represents one of the most reliable sources for providing a sufficient yield of cells in a good quality. Herein, used bone marrow processing kits were evaluated as a valuable source of MSCs. Methods: Bone marrow mononuclear cells (MNCs) were recovered from used bone marrow processing kits after routine bone marrow processing by using the COBE 2991 Cell Processor (CaridianBCT Inc.). The MSCs were isolated from the recovered MNCs using a standard plastic adherence method. Immunophenotyping and differentiation assays were performed to clarify the characteristics of the isolated MSCs. Results: An average of $1{\times}10^8$ bone marrow MNCs was collected, and the MSCs were successfully isolated from the recovered bone marrow MNCs in all case. The isolated MSCs were positive for essential MSC surface molecules (CD29, CD44, CD73, CD90, CD105) and they were negative for most hematopoietic and endothelial cell markers (CD34, CD45, CD31, CD14). The isolated MSCs were capable of differentiation along the osteogenic, adipogenic and chondrogenic pathways. Conclusion: MSCs isolated from used bone marrow processing kits are an alternative and ethical source of bone marrow derived MSCs, and they can be used for research purposes.

키워드

과제정보

연구 과제 주관 기관 : 대한수혈학회

참고문헌

  1. Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103:697-705 https://doi.org/10.1172/JCI5298
  2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-7 https://doi.org/10.1126/science.284.5411.143
  3. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528-30 https://doi.org/10.1126/science.279.5356.1528
  4. da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 2006;119:2204-13 https://doi.org/10.1242/jcs.02932
  5. Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WW, Gordon PL, Neel M, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309-13 https://doi.org/10.1038/6529
  6. Bieback K, Kern S, Kluter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 2004;22:625-34 https://doi.org/10.1634/stemcells.22-4-625
  7. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279-95 https://doi.org/10.1091/mbc.E02-02-0105
  8. In 't Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, Fibbe WE, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004;22:1338-45 https://doi.org/10.1634/stemcells.2004-0058
  9. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100-6 https://doi.org/10.1038/nbt1274
  10. Kemp KC, Hows J, Donaldson C. Bone marrow-derived mesenchymal stem cells. Leuk Lymphoma 2005;46:1531-44 https://doi.org/10.1080/10428190500215076
  11. D'Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res 1999;14:1115-22 https://doi.org/10.1359/jbmr.1999.14.7.1115
  12. Hung SC, Chen NJ, Hsieh SL, Li H, Ma HL, Lo WH. Isolation and characterization of size-sieved stem cells from human bone marrow. Stem Cells 2002;20:249-58 https://doi.org/10.1634/stemcells.20-3-249
  13. Cheng SL, Yang JW, Rifas L, Zhang SF, Avioli LV. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 1994;134:277-86 https://doi.org/10.1210/en.134.1.277
  14. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999;107:275-81 https://doi.org/10.1046/j.1365-2141.1999.01715.x
  15. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41-9 https://doi.org/10.1038/nature00870
  16. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276: 71-4 https://doi.org/10.1126/science.276.5309.71
  17. Mageed AS, Pietryga DW, DeHeer DH, West RA. Isolation of large numbers of mesenchymal stem cells from the washings of bone marrow collection bags: characterization of fresh mesenchymal stem cells. Transplantation 2007;83:1019-26 https://doi.org/10.1097/01.tp.0000259752.13304.0b
  18. Dvorakova J, Hruba A, Velebny V, Kubala L. Isolation and characterization of mesenchymal stem cell population entrapped in bone marrow collection sets. Cell Biol Int 2008;32: 1116-25 https://doi.org/10.1016/j.cellbi.2008.04.024
  19. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315-7 https://doi.org/10.1080/14653240600855905