[국내논문]세포 크기 차이를 이용한 유세포 분석을 통한 인간배아줄기세포 유래 기능성 혈관세포의 확립 Establishment of Functional Cells for Vascular Defect Disease from Human Embryonic Stem Cell via Region Sorting Depending on Cell Volume원문보기
인간배아줄기세포는 인간배아줄기세포가 가지는 전 분화능 등의 특이적 특성으로 인해 재생의학 분야에서 세포 치료제의 근원으로 널리 각광받고 있다. 그러나, 미분화 상태의 인간배아줄기세포를 세포치료제로 이용하기 위해서는 인간배아줄기세포 주 유래 기능성 세포를 확립이 반드시 요구된다. 본 연구에서는, 미분화 상태의 인간배아줄기세포주로부터 기능성 세포의 확립을 위해, 혈관계통의 세포로 분화를 유도하였으며, 분화 유도 후 세포의 크기 차이를 이용하여 특정 세포군 만을 분리하여 그 기능성을 비교 분석하였다. 그 결과, VEGF를 이용하여 분화 시킨 세포군에서 약 10%의 PECAM 양성 세포군을 확인할 수 있었으며, 분리 및 세포 이식을 위해 세포를 단일 세포군으로 만들었다. 단일 세포군의 형성 후, 유세포 분석기를 이용한 세포 분리 기법을 이용하여 FCS를 기준으로 한 세포 크기의 차이를 이용하여 특정 세포군 만을 분리하여, 하지 허혈 동물 모델로의 이식을 통해, 비 분리 세포군과 치료 효능을 비교 분석을 실시하였다. 세포 이식 4주 후, 혈류량 복구율이 FSC 기준 분리 군의 경우 54%, 비 분리군의 경우 17%를 보이는 것을 확인하였다. 이 결과는, 초기 분화 유도 후 세포 크기차이를 이용한 세포 분리법이 기능성 세포 획득에 이용될 수 있음을 시사한다. 이와 같은 방법을 통해 다양한 종류의 기능성 세포 분리에 이용될 수 있을 것이라 생각된다.
인간배아줄기세포는 인간배아줄기세포가 가지는 전 분화능 등의 특이적 특성으로 인해 재생의학 분야에서 세포 치료제의 근원으로 널리 각광받고 있다. 그러나, 미분화 상태의 인간배아줄기세포를 세포치료제로 이용하기 위해서는 인간배아줄기세포 주 유래 기능성 세포를 확립이 반드시 요구된다. 본 연구에서는, 미분화 상태의 인간배아줄기세포주로부터 기능성 세포의 확립을 위해, 혈관계통의 세포로 분화를 유도하였으며, 분화 유도 후 세포의 크기 차이를 이용하여 특정 세포군 만을 분리하여 그 기능성을 비교 분석하였다. 그 결과, VEGF를 이용하여 분화 시킨 세포군에서 약 10%의 PECAM 양성 세포군을 확인할 수 있었으며, 분리 및 세포 이식을 위해 세포를 단일 세포군으로 만들었다. 단일 세포군의 형성 후, 유세포 분석기를 이용한 세포 분리 기법을 이용하여 FCS를 기준으로 한 세포 크기의 차이를 이용하여 특정 세포군 만을 분리하여, 하지 허혈 동물 모델로의 이식을 통해, 비 분리 세포군과 치료 효능을 비교 분석을 실시하였다. 세포 이식 4주 후, 혈류량 복구율이 FSC 기준 분리 군의 경우 54%, 비 분리군의 경우 17%를 보이는 것을 확인하였다. 이 결과는, 초기 분화 유도 후 세포 크기차이를 이용한 세포 분리법이 기능성 세포 획득에 이용될 수 있음을 시사한다. 이와 같은 방법을 통해 다양한 종류의 기능성 세포 분리에 이용될 수 있을 것이라 생각된다.
Human embryonic stem cells have been highlighted as a valuable cellular source in the regenerative medicine field, due to their pluripotency. However, there is the challenge of the establishment of specific functional cell type forms of undifferentiated human embryonic stem cells (hESC). To establis...
Human embryonic stem cells have been highlighted as a valuable cellular source in the regenerative medicine field, due to their pluripotency. However, there is the challenge of the establishment of specific functional cell type forms of undifferentiated human embryonic stem cells (hESC). To establish and purify functional cell types from hESCs, we differentiated undifferentiated hESCs into vascular lineage cells and sorted the specific cell population from the whole cell population, depending on their cell volume, and compared them with the non-sorted cell population. We observed that about 10% of the PECAM positive population existed in the VEGF induced differentiating human embryoid body (hEB), and differentiated hEBs were made into single cells for cell transplantation. After making single cells, we performed cell sorting using a fluorescence-activated cell sorter (FACs), according to their cell volume on the basis of FSC region gating, and compared their therapeutic capacity with the non-sorted cell population through cell transplantation into hindlimb ischemic disease model mice. 4 Weeks after cell transplantation, the recovery rate of blood perfusion reached 54% and 17% in the FSC regions of sorted cells- and non-sorted cells, respectively. This result suggests that derivation of a functional cell population from hESCs can be performed through cell sorting on the basis of cell volume after preliminary differentiation induction. This approach may then greatly contribute to overcoming the limitations of marker sorting.
Human embryonic stem cells have been highlighted as a valuable cellular source in the regenerative medicine field, due to their pluripotency. However, there is the challenge of the establishment of specific functional cell type forms of undifferentiated human embryonic stem cells (hESC). To establish and purify functional cell types from hESCs, we differentiated undifferentiated hESCs into vascular lineage cells and sorted the specific cell population from the whole cell population, depending on their cell volume, and compared them with the non-sorted cell population. We observed that about 10% of the PECAM positive population existed in the VEGF induced differentiating human embryoid body (hEB), and differentiated hEBs were made into single cells for cell transplantation. After making single cells, we performed cell sorting using a fluorescence-activated cell sorter (FACs), according to their cell volume on the basis of FSC region gating, and compared their therapeutic capacity with the non-sorted cell population through cell transplantation into hindlimb ischemic disease model mice. 4 Weeks after cell transplantation, the recovery rate of blood perfusion reached 54% and 17% in the FSC regions of sorted cells- and non-sorted cells, respectively. This result suggests that derivation of a functional cell population from hESCs can be performed through cell sorting on the basis of cell volume after preliminary differentiation induction. This approach may then greatly contribute to overcoming the limitations of marker sorting.
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가설 설정
Therefore, we hypothesized that cell sorting on the basis of their cell size could be one option for purifying specific cell type, to overcome the limitation of low percentage of marker positive cell population, after preliminary differentiation induction, because cell size will be more similar after preliminary induction. Consequently, this study focuses on FSC region sorting depending on cell volume after preliminary differentiation induction using representative vascular lineage differentiation induction factor, VEGF [7], to enhance the differentiation efficiency of hESCs into vascular lineage cells.
Here, we hypothesized that cell sorting on the basis of their cell size could be one option for purifying specific cell type, to overcome the limitation of low percentage of marker positive cell population and low cell viability after laser cell sorting. And resulted cell product shown the higher possibility as cellular source for cell therapy in vascular defective disease field.
제안 방법
Therefore, we hypothesized that cell sorting on the basis of their cell size could be one option for purifying specific cell type, to overcome the limitation of low percentage of marker positive cell population, after preliminary differentiation induction, because cell size will be more similar after preliminary induction. Consequently, this study focuses on FSC region sorting depending on cell volume after preliminary differentiation induction using representative vascular lineage differentiation induction factor, VEGF [7], to enhance the differentiation efficiency of hESCs into vascular lineage cells.
Mice were monitored by serial scann-ing of surface blood flow in hindlimbs at several time points over a period of 4 weeks. The digital color-coded images were analyzed to quantify the blood flow in the region from the knee joint to the toe, and the mean values of perfusion were calculated.
Morphologically, sorted cells were more uniformed than non-sorted cells, and cells were culture on matorigel to compare the functional characteristics of both cell population as vascular cells, thinner and un-uniformed structure were observed in non-sorted cells, on the other hand, sorted cell population spontaneously organized into vessel-like capillary structures on matrigel within 24 hours and maintained them. Also, ac-LDL uptake analysis, the fluorescence of ac-LDL which detected by FACs were recorded as 21.
대상 데이터
All images were analyzed with a LSM 510 META confocal microscope (Carl Zeiss Inc.; Oberkochen, Germany).
데이터처리
Quantitative data are expressed as mean±SD. Statistical analysis was performed with the t-test. A value of P<0.
성능/효과
Also, through this study, we could confirmed that the possibility of FSC region gating cell sorting method for generating functional cellular source from hESCs.
참고문헌 (27)
Ahn, S. E., S. Kim, K. H. Park, S. H. Moon, H. J. Lee, G. J. Kim et al. 2006. Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells. Biochem. Biophy. Res. Co. 340: 403- 408.
Amit, M., M. K. Carpenter, M. S. Inokuma, C. P. Chiu, C. P. Harris, M. A. Waknitz et al. 2000. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227: 271-278.
Asahara, T., A. Kawamoto, and H. Masuda. Concise review: circulating endothelial progenitor cells for vascular medicine. Stem Cells 29: 1650-1655.
Asahara, T., T. Murohara, A. Sullivan, M. Silver, R. van der Zee, T. Li et al. 1997. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964-967.
Boyd, N. L., S. K. Dhara, R. Rekaya, E. A. Godbey, K. Hasneen, R. R. Rao et al. 2007. BMP4 promotes formation of primitive vascular networks in human embryonic stem cellderived embryoid bodies. Exp. Biol. Med. (Maywood) 232: 833-843.
Byrne, A. M., B.-H. D., Harmey J. H. 2005 Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J. Cell. Mol. Med. 9: 777-794.
Cho, S. W., S. H. Moon, S. H. Lee, S. W. Kang, J. Kim, J. M. Lim et al. 2007. Improvement of postnatal neovascularization by human embryonic stem cell derived endotheliallike cell transplantation in a mouse model of hindlimb ischemia. Circulation 116: 2409-2419.
Drukker, M., H. Katchman, G. Katz, S.Even-Tov. Friedman, E. Shezen, E. Hornstein et al. 2006. Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells. 24: 221- 229.
Dvash, T., D. Ben-Yosef, and R. Eiges. 2006. Human embryonic stem cells as a powerful tool for studying human embryogenesis. Pediatr. Res. 60: 111-117.
Ferreira, L. S., S. Gerecht, J. Fuller, H. F. Shieh, G. Vunjak- Novakovic, and R. Langer. 2007. Bioactive hydrogel scaffolds for controllable vascular differentiation of human embryonic stem cells. Biomaterials 28: 2706-2717.
Kim, J., S. H. Moon, S. H. Lee, D. R. Lee, G. Y. Koh, and H. M. Chung. 2007. Effective isolation and culture of endothelial cells in embryoid body differentiated from human embryonic stem cells. Stem Cells Dev. 16: 269-280.
Klimanskaya, I., Y. Chung, S. Becker, S. J. Lu, and R. Lanza. 2006. Human embryonic stem cell lines derived from single blastomeres. Nature 444: 481-485.
Layman, H., M. Sacasa, A. E. Murphy, A. M. Murphy, S. M. Pham, and F. M. Andreopoulous. 2008 Co-delivery of FGF-2 and G-CSF from gelatin-based hydrogels as angiogenic therapy in a murine critical limb ischemic model.Co-delivery of FGF-2 and G-CSF from gelatin-based hydrogels as angiogenic therapy in a murine critical limb ischemic model. Acta Biomater. 5: 230-239.
Levenberg, S., J. S. Golub, M. Amit, J. Itskovitz-Eldor, and R. Langer. 2002. Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U S A 99: 4391- 4396.
Loges, S., C. Roncal, and P. Carmeliet. 2009. Development of targeted angiogenic medicine. J. Thromb. Haemost. 7: 21- 33.
Lu, S. J., Q. Feng, S. Caballero, Y. Chen, M. A. Moore, M. B. Grant et al. 2007. Generation of functional hemangioblasts from human embryonic stem cells. Nat. Methods 4: 501-509.
Mr, L. 1999. Immunofluorescence Techniques in Flow Cytometry and Sorting Wiley: 341-353.
Ornatsky, O., D. Bandura, V. Baranov, M. Nitz, M. A. Winnik, and S. Tanner. Highly multiparametric analysis by mass cytometry. J. Immunol. Methods 361: 1-20.
Qian, Y., C. Wei, E.-H. Lee, F. Campbell, J. Halliley, J. Lee et al. 2010. Elucidation of seventeen human peripheral blood B-cell subsets and quantification of the tetanus response using a density-based method for the automated identification of cell populations in multidimensional flow cytometry data. Cytometry Part B: Clinical Cytometry 78B.
Thomson, J. A., J. Itskovitz-Eldor, S. S. Shapiro, M. A. Waknitz, J. J. Swiergiel, V. S. Marshall et al. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282: 1145-1147.
Wang, L., L. Li, F. Shojaei, K. Levac, C. Cerdan, P. Menen dez et al. 2004. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity 21: 31-41.
Wang, Z. Z., P. Au, T. Chen, Y. Shao, L. M. Daheron, H. Bai et al. 2007. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nat Biotechnol. 25: 317-318.
Yamahara, K., M. Sone, H. Itoh, J. K. Yamashita, T. Yurugi- Kobayashi, K. Homma et al. 2008. Augmentation of neovascularizaiton in hindlimb ischemia by combined transplantation of human embryonic stem cells-derived endothelial and mural cells. PLoS ONE 3: e1666.
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