독시사이클린에 의한 사람 배아 줄기세포 및 유도 만능 줄기세포에서의 효율적인 자가증식과 전분화능 유지 효과

Abstract

사람배아줄기세포[human embryonic stem cells (hESCs)] 및 사람유도만능줄기세포[induced pluripotent stem cells (iPSCs)]의 배양은 값이 비싼 인자들과 사이토카인들을 포함하는 배양배지를 매일 갈아주어야 함으로 세포를 유지하는 데에 많은 비용과 노동력을 필요로 한다. 선행연구에서 사람전분화줄기세포(human pluripotent stem cell; hESCs/hiPSCs)의 생존과 자가재생 촉진에 독시사이클린의 처리가 효과가 있음을 확인하였고, 따라서 본 연구에서는 사람전분화 줄기세포의 배양시에 비용을 절감하며 적은 노동으로 보다 실용적인 세포 배양을 가능하게 하는 독시사이클린의 효과에 주목하였다. 세포의 상태와 관계없이 독시사이클린을 첨가하지 않은 배지를 사용한 사람전분화줄기세포는 3일간 배양배지를 갈지 않았을 때 세포가 모두 죽었으나, 독시사이클린을 처리한 세포는 최소 3일간 배지를 갈지 않아도 세포의 생존이나 증식이 여전히 가능함을 확인하였다. 따라서, 사람전분화 줄기세포의 배양시 독시사이클린의 첨가를 통해 3일 단위로 배양배지를 갈아주어도 세포특성을 잃지 않고, 세포의 자가증식 및 전분화능을 유지하며 배양이 가능하며, 이는 독시사이클린의 첨가가 매일 배양배지를 갈아 주는 번거로움을 덜어주며 값이 비싼 배양배지의 사용량을 3분의 1까지도 줄일 수 있음을 확인할 수 있었다.|Culturing human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) is one of the most costly and labor-intensive tissue cultures, as media containing expensive factors and cytokines should be changed every day to maintain and propagate undifferentiated hESCs/iPSCs in vitro. We recently reported that doxycycline, an anti-bacterial agent, had dramatic effects on hESC/iPSC survival and promoted self-renewal. In this study, we extended the effects of doxycycline to a more practical issue to save cost and labor in hESC/iPSC cultures. Regardless of cultured cell conditions, hESCs/iPSCs in doxycycline-supplemented media were viable and proliferating for at least 3 days without media change, while none or few viable cells were detected in the absence of doxycycline in the same conditions. Thus, hESCs/iPSCs supplemented with doxycycline can be cultured for a long period of time with media changes at 3-day intervals without altering their self-renewal and pluripotent properties, indicating that doxycycline supplementation can reduce the frequency of media changes and the amount of media required by 1/3. These findings strongly encourage the use of doxycycline to save cost and labor in culturing hESCs/iPSCs.; Culturing human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) is one of the most costly and labor-intensive tissue cultures, as media containing expensive factors and cytokines should be changed every day to maintain and propagate undifferentiated hESCs/iPSCs in vitro. We recently reported that doxycycline, an anti-bacterial agent, had dramatic effects on hESC/iPSC survival and promoted self-renewal. In this study, we extended the effects of doxycycline to a more practical issue to save cost and labor in hESC/iPSC cultures. Regardless of cultured cell conditions, hESCs/iPSCs in doxycycline-supplemented media were viable and proliferating for at least 3 days without media change, while none or few viable cells were detected in the absence of doxycycline in the same conditions. Thus, hESCs/iPSCs supplemented with doxycycline can be cultured for a long period of time with media changes at 3-day intervals without altering their self-renewal and pluripotent properties, indicating that doxycycline supplementation can reduce the frequency of media changes and the amount of media required by 1/3. These findings strongly encourage the use of doxycycline to save cost and labor in culturing hESCs/iPSCs.