KR20180023641A - Method for enhancing reprogramming efficiency of induced plutipotent stem cells from somatic cells by long term cell culture post-irradiation - Google Patents
Method for enhancing reprogramming efficiency of induced plutipotent stem cells from somatic cells by long term cell culture post-irradiation Download PDFInfo
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Abstract
Description
본 발명은 방사선 조사 후 장기 세포배양에 의한 유도만능줄기세포 리프로그래밍 효율 증진 방법에 관한 것이다.The present invention relates to a method for promoting induced pluripotent stem cell reprogramming efficiency by long-term cell culture after irradiation.
분화가 끝난 체세포를 미분화 상태의 세포 (e.g., 줄기세포)로 되돌리는 과정을 리프로그래밍(reprogramming)으로 명명하고 있으며, 현재까지 체세포 핵 치환 (somatic cell nuclear transfer, SCNT), 유도만능줄기세포 리프로그래밍 (induced pluripotent stem cell reprogramming, iPS reprogramming), 세포융합 (cell fusion) 방법이 보고되고 있다. The process of returning differentiated somatic cells to undifferentiated cells (eg, stem cells) has been termed reprogramming. To date, somatic cell nuclear transfer (SCNT), induced pluripotent stem cell reprogramming induced pluripotent stem cell reprogramming (iPS) reprogramming, and cell fusion.
역분화줄기세포는 유도만능줄기세포로 지칭되며, 분화가 끝난 체세포에 역분화인자들을 삽입하여 분화이전의 세포단계로 되돌린 세포로 배아줄기세포처럼 자기재생 및 만능분화능을 가지고 있어 차세대 줄기세포치료제로서 급부상하고 있지만, 낮은 생성 효율로 인해 임상적용에 어려움을 겪고 있는 실정이다.Stem cells are called inducible pluripotent stem cells. They are cells that have been reintroduced into the cell stage prior to differentiation by inserting de-differentiation factors into the differentiated somatic cells. They have self-renewal and universal differentiation potentials like embryonic stem cells, , But the clinical application is difficult due to the low production efficiency.
이러한 낮은 유도만능줄기세포 리프로그래밍 효율을 높이기 위해 다양한 인자 등이 보고되었으나, 방사선 조사 후 (post-irradiation) 리프로그래밍 효율 증진에 대한 보고는 미비하다.Various factors have been reported to increase the efficiency of such low induced pluripotent stem cell reprogramming, but there is little report on post-irradiation reprogramming efficiency enhancement.
본 발명의 목적은 방사선 조사 후 장기 세포배양에 의한 유도만능줄기세포 리프로그래밍 효율 증진 방법을 제공하는데 있다.It is an object of the present invention to provide a method for promoting induced pluripotent stem cell reprogramming efficiency by long-term cell culture after irradiation.
본 발명은 (1) 섬유아세포에 방사선을 조사하는 단계; (2) 상기 방사선이 조사된 섬유아세포를 10 내지 25일 배양하는 단계; 및 (3) 상기 배양된 섬유아세포를 유도만능줄기세포로 리프로그래밍시키는 단계를 포함하는 방사선 조사 후 장기 세포배양에 의한 유도만능줄기세포 리프로그래밍 효율 증진 방법을 제공한다.(1) irradiating fibroblasts with radiation; (2) culturing the irradiated fibroblasts for 10 to 25 days; And (3) reprogramming the cultured fibroblasts to inducible pluripotent stem cells. The present invention further provides a method for promoting induced pluripotent stem cell reprogramming.
본 발명은 방사선 조사 후 장기 세포배양에 의한 유도만능줄기세포 리프로그래밍 효율 증진 방법을 제공한다. 본 발명에 따르면 방사선이 조사된 세포를 다양한 요일별로 배양 후 유도만능줄기세포로의 리프로그래밍 효율을 조사한 결과, 3일째 배양된 세포는 유도만능줄기세포 생성율이 감소한 반면 14일째 배양된 세포에서는 유도만능줄기세포 생성이 현저히 증가됨을 관찰할 수 있었다. 또한 고효율로 리프로그램된 세포들에서 줄기세포 특성 분석을 통해 유도만능줄기세포임을 확인하여 방사선 조사 후 장기배양된 세포는 유도만능줄기세포 리프로그래밍이 고효율로 증진됨을 증명하였다. 따라서, 본 발명에 적용된 방사선 조사에 의한 유도만능줄기세포 리프로그래밍 증진법은 유도만능줄기세포 생성 효율 증진인자 탐색 및 기전 연구에 활용될 수 있을 것이다.The present invention provides a method for promoting induced pluripotent stem cell reprogramming efficiency by long-term cell culture after irradiation. According to the present invention, the reprogramming efficiency of inducing pluripotent stem cells after culturing the irradiated cells by various days of days showed that induction of pluripotent stem cells was decreased in the cells cultured on day 3, And the production of stem cells was remarkably increased. In addition, we confirmed that the cells were induced pluripotent stem cells through the characterization of stem cells in high - reprogrammed cells. Thus, long - term cultured cells after irradiation showed that inducible pluripotent stem cell reprogramming was promoted with high efficiency. Therefore, the induced pluripotent stem cell reprogramming promoted by radiation irradiation applied to the present invention can be utilized for research on the inducible pluripotent stem cell production promoting factor and its mechanism.
도 1은 방사선 조사된 섬유아세포에서 유도만능줄기세포 리프로그래밍 실험 모식도(A) 및 섬유아세포에서 리프로그램된 세포(B)를 나타낸다.
도 2는 방사선 조사 후 장기배양된 섬유아세포에서 증가된 유도만능줄기세포 리프로그래밍 효율을 나타낸다.
도 3 및 도 4는 방사선 조사 후 장기배양된 세포로부터 리프로그램된 세포들의 만능줄기세포 특성 분석 결과를 나타낸다.Figure 1 shows a schematic diagram (A) of induced pluripotent stem cell reprogramming in irradiated fibroblasts and a reprogrammed cell (B) in fibroblasts.
Fig. 2 shows the induced pluripotent stem cell reprogramming efficiency in fibroblasts cultured after irradiation.
FIG. 3 and FIG. 4 show the results of analysis of pluripotent stem cell characteristics of the reprogrammed cells from the cells cultured for a long time after irradiation.
본 발명은 (1) 섬유아세포에 방사선을 조사하는 단계; (2) 상기 방사선이 조사된 섬유아세포를 10 내지 25일 배양하는 단계; 및 (3) 상기 배양된 섬유아세포를 유도만능줄기세포로 리프로그래밍시키는 단계를 포함하는 방사선 조사 후 장기 세포배양에 의한 유도만능줄기세포 리프로그래밍 효율 증진 방법을 제공한다. (1) irradiating fibroblasts with radiation; (2) culturing the irradiated fibroblasts for 10 to 25 days; And (3) reprogramming the cultured fibroblasts to inducible pluripotent stem cells. The present invention further provides a method for promoting induced pluripotent stem cell reprogramming.
바람직하게는, 상기 방사선 조사는 1 내지 5Gy 단일 방사선으로 조사할 수 있으나, 이에 한정되는 것은 아니다.Preferably, the radiation is irradiated by a single radiation of 1 to 5 Gy, but is not limited thereto.
바람직하게는, 상기 (2) 단계의 배양은 37℃에서 14일 동안 배양할 수 있으나, 이에 한정되는 것은 아니다.Preferably, the culture in step (2) above can be cultured at 37 DEG C for 14 days, but is not limited thereto.
바람직하게는, 상기 (3) 단계 리프로그래밍은 섬유아세포에 리프로그래밍 인자인 Klf4, Oct4, Sox2 및 c-Myc를 도입할 수 있으나, 이에 한정되는 것은 아니다. Preferably, the step (3) reprogramming may include, but is not limited to, introducing fibroblast reprogramming factors Klf4, Oct4, Sox2 and c-Myc.
본 발명에 있어서, "리프로그래밍(reprogramming)"은 분화가 끝난 체세포를 미분화 상태의 세포 (e.g., 줄기세포)로 되돌리는 과정을 의미한다.In the present invention, "reprogramming" refers to a process of returning differentiated somatic cells to undifferentiated cells (e.g., stem cells).
본 발명에 있어서, "유도만능줄기세포(induced pluripotent stem cell; iPSC)"는 분화가 끝난 체세포에 역분화인자들을 삽입하여 분화 이전의 세포 단계로 되돌린 세포로 배아줄기세포처럼 자기재생과 만능분화능을 가진 줄기세포이다.In the present invention, "induced pluripotent stem cell (iPSC)" is a cell that has been transformed into a cell stage prior to differentiation by inserting dedifferentiation factors into the differentiated somatic cells. Like embryonic stem cells, .
이하에서는, 본 발명을 한정하지 않는 실시예에 따라 본 발명을 상세히 설명한다. 본 발명의 하기 실시예는 본 발명을 구체화하기 위한 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아님은 물론이다. 따라서, 본 발명의 상세한 설명 및 실시예로부터 본 발명이 속하는 기술분야의 전문가가 용이하게 유추할 수 있는 것은 본 발명의 권리범위에 속하는 것으로 해석된다. Hereinafter, the present invention will be described in detail with reference to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
<< 실험예Experimental Example >>
하기의 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다.The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.
1. 인체 섬유아세포 및 1. Human fibroblasts and 유도만능줄기세포Induced pluripotent stem cells 배양 culture
ATCC로부터 분양받은 NUFF (인체섬유아세포)을 10% 혈청이 함유된 DMEM (Giboco BRL., USA) 성장배지에 넣고 37℃, 5% CO2 incubator에서 배양하였다. 배지는 3일에 1번 교체하였으며, 세포 밀도가 80% 되었을 때 계대 배양하였다. 섬유아세포에 5Gy 단일 방사선 조사하였다. 생성된 유도만능줄기세포는 matrigel coated plate에 1:6로 분주후 10μM ROCK 저해제가 함유된 mTeSR1 medium (Stemcell Technology Inc., USA) 첨가하고 37℃, 5% CO2 조건하에서 배양하였다. 다음 날 배지를 교환 후 2일에 한 번씩 배지를 교체해준다.NUFF (human fibroblast) from ATCC was cultured in DMEM (Gibco BRL, USA) containing 10% serum at 37 ° C and 5% CO 2 incubator. The medium was changed once every 3 days and subcultured when cell density was 80%. Fibroblasts were irradiated with 5Gy single radiation. The resulting induced pluripotent stem cells were mixed with matrigel coated plates at a ratio of 1: 6, and then mTeSR1 medium (Stemcell Technology Inc., USA) containing 10 μM ROCK inhibitor was added and cultured at 37 ° C and 5% CO 2 . Replace the medium the next day and replace the medium once every two days.
2. 2. 리프로그래밍인자Reprogramming factor 레트로 바이러스Retrovirus 생성 produce
gp293 (Clontec Inc., USA) 세포를 100mm dish에 6x106으로 분주하여 5% CO2 incubator에서 24시간 배양하였다. 다음 날, gp293세포에 lipofectamin 2000 kit 매뉴얼에 따라, 각각의 1㎍/ml 리프로그래밍인자들 (Klf4, Oct4, Sox2, c-Myc) 과 0.5㎍/ml VSVG (바이러스 envelope vector) 와 co-transfection하고 37℃, 5% CO2 incubator에서 24시간 배양한 후 10% DMEM으로 교체하였다. 배양 2일차 및 3일차에 상등액을 모아 0.45mm filter로 여과하여 리프로그래밍 인자들이 함유된 바이러스를 생산하였다.gp293 (Clontec Inc., USA) cells were seeded in a 100 mm dish at 6 × 10 6 and cultured in a 5% CO 2 incubator for 24 hours. The next day, gp293 cells were co-transfected with 1 μg / ml reprogramming factors (Klf4, Oct4, Sox2, c-Myc) and 0.5 μg / ml VSVG (viral envelope vector) according to the lipofectamine 2000 kit manual After incubation for 24 h at 37 ° C in a 5% CO 2 incubator, the cells were replaced with 10% DMEM. On the 2nd and 3rd day of culture, the supernatant was collected and filtered with a 0.45 mm filter to produce a virus containing reprogramming factors.
3. 방사선조사 후 배양된 3. After irradiation, 인체섬유아세포에서In human fibroblasts 유도만능줄기세포로의Induced pluripotent stem cells 리프로그래밍Reprogramming
바이러스 생성 하루 전 대조군 또는 실험군 섬유아세포 (방사선 조사 후 표시된 요일 동안 배양된 세포)를 5x104cells/well (6 well plate)로 부착 후 리프로그래밍인자 바이러스 와 5㎍/㎖ polybrene이 첨가된 배지로 배양하였다. 5일 동안 형질 전환된 섬유아세포를 1x104cell/well이 되게 Matrigel coated plate에 분주하고 5% CO2 incubator에서 24시간 배양하였다. 다음날, human iPS cell (TeSR-E7 media, Stemcell Technology Inc.) 유도 배지로 교체해주고, 만능줄기세포인 배아줄기세포 유사 콜로니 (hES-like coloies)가 나타날 때 까지 매일 배지를 교체해주었다.Cells were incubated with 5 × 10 4 cells / well (6-well plates) in a control or experimental group of fibroblasts (cells cultured for the days indicated after irradiation), and then cultured in media supplemented with reprogramming factor virus and 5 μg / ml polybrene Respectively. Transfected fibroblasts were plated on Matrigel coated plates at 1 × 10 4 cells / well for 5 days and cultured in a 5% CO 2 incubator for 24 hours. The next day, the medium was replaced with a human iPS cell (TeSR-E7 media, Stemcell Technology Inc.) induction medium and the medium was changed daily until the expression of pluripotent embryonic stem cells (hES-like coloies).
4. 4. 인체섬유아세포의Human fibroblast 리프로그래밍Reprogramming 효율 분석 Efficiency analysis
리프로그램된 세포를 R-phycoerythrin (R-PE)이 결합된 SSEA4 (1:500 dilution, Stemgent, USA) 와 fluorescein isothiocyanate (FITC)이 결합된 Tra1-81 (1:100 dilution, stemgent, USA)가 포함된 배양 배지에서 37℃, 5% CO2 incubator에서 1시간 염색 후 형광현미경으로 관찰하였다. SSEA4는 red color로 관찰하였으며, Tra1-81은 green color로 관찰하고 염색된 콜로니 수를 카운터하여 정량화 하였다. 또한 Alkaline Phosphatase Staining kit 매뉴얼에 따라 (System Biosciences, USA), 리프로그램된 세포를 PBS로 2회 세척하고, 4% paraformaldehyde로 고정한 후, Blue-color AP 용액으로 염색하고 PBS로 2회 세척한 후 콜로니 염색 여부를 광학현미경 아래서 관찰하였다. 염색된 콜로니 수를 카운터하여 정량화 하였다. (1: 100 dilution, stemgent, USA) in which R-phycoerythrin (R-PE) conjugated SSEA4 (1: 500 dilution, Stemgent, USA) and fluorescein isothiocyanate Were stained in a 5% CO 2 incubator at 37 ° C for 1 hour and then observed with a fluorescence microscope. SSEA4 was observed in red color, Tra1-81 was observed in green color, and the number of stained colonies was counted and quantified. In addition, the reprogrammed cells were washed twice with PBS, fixed with 4% paraformaldehyde, stained with Blue-color AP solution, washed twice with PBS according to the Alkaline Phosphatase Staining kit manual (System Biosciences, USA) Dyeing was observed under an optical microscope. The number of stained colonies was counted and quantified.
5. 면역염색5. Immunostaining
리프로그램된 세포들을 4% paraformaldehyde로 상온에서 20min 고정한다. 고정된 세포를 1% BSA 및 0.5% Triton X-100인 함유된 PBS로 상온에서 1시간 반응시킨 후 각각의 일차 항체들 Oct4 (1:100, SantaCruz, CA, USA), Sox2 (1:100, Cell Signalling, Danvers, MA, USA), Nanog (1:200, Cosmo Bio, Koto-Ku, Japan) 처리하고, FITC-conjugated goat anti-rabbit IgG 또는 anti-mouse IgG (1:100, Invitrogen, Carlsbad, CA)를 이차항체로 반응하였다. 형광이미지는 형광현미경 (Olympus, Shinjuku, Tokyo, Japan)으로 분석하였다. DAPI를 핵염색 용액으로 사용하였다. Reprogrammed cells are fixed with 4% paraformaldehyde at room temperature for 20 min. The immobilized cells were incubated with PBS containing 1% BSA and 0.5% Triton X-100 for 1 hour at room temperature. Each primary antibody, Oct4 (1: 100, SantaCruz, CA, USA), Sox2 Conjugated goat anti-rabbit IgG or anti-mouse IgG (1: 100, Invitrogen, Carlsbad, CA), Nanog (1: 200, Cosmo Bio, Koto- CA) were reacted with secondary antibodies. Fluorescence images were analyzed by fluorescence microscopy (Olympus, Shinjuku, Tokyo, Japan). DAPI was used as a nuclear staining solution.
6. 6. qPCRqPCR
섬유아세포와 리프로그램된 세포에서 RNA minikit (Qiagen, Inc.)를 이용해 total RNA를 추출한 다음 Accupower RT mix reagent (Bioneer Corp., Seoul,Korea)를 사용해 cDNA로 만들었다. 실시간 PCR (Real-time PCR) FastStart Essential DNA Green Master (Roche, Indianapolis, IN, USA)를 사용하여 수행하였다. 본 발명에 사용된 프라이머 서열은 표 1과 같다.Total RNA was extracted from fibroblasts and reprogrammed cells using RNA minikit (Qiagen, Inc.) and then made into cDNA using Accupower RT mix reagent (Bioneer Corp., Seoul, Korea). Real-time PCR was performed using FastStart Essential DNA Green Master (Roche, Indianapolis, IN, USA). The primer sequences used in the present invention are shown in Table 1.
<< 실시예Example 1> 방사선 조사 후 장기 배양된 섬유아세포에서 1> Long-term culture of fibroblasts after irradiation 증가된Increased 유도만능줄Guidewire 기세포 리프로그래밍 효율 확인Checking programming efficiency
방사선 조사된 섬유아세포에서 유도만능줄기세포 리프로그래밍 실험 모식도를 도 1A에 나타냈다. 대조군과 방사선 조사된 처리군을 리프로그래밍 전사인자들을 이용하여 리프로그램 후 시간에 따른 배아줄기세포세포 유사 콜로니 모양 및 줄기세포 특이적 표면마커 (SSEA4 및 Tra1-81)을 2주째부터 관찰할 수 있었다(도 1B).A schematic diagram of an induced pluripotent stem cell reprogramming experiment in irradiated fibroblasts is shown in Fig. 1A. Using the reprogramming transcription factors in the control and irradiated groups, embryonic stem cell-like colony shape and stem cell specific surface markers (SSEA4 and Tra1-81) were observed from the 2nd week after reprogramming (Fig. 1B).
5Gy 방사선을 조사 후 3일, 7일 및 14일 배양된 세포에 리프로그래밍 인자들을 형질도입 후 배아줄기세포 유사 콜로니 형태로 Tra1-81 (유도만능줄기세포 표면마커) 항체가 염색된 콜로니 개수를 counting 결과 방사선 조사 후 장기배양된 세포에서 약 2배이상 리프로그래밍 효율이 증가됨을 관찰할수 있었다(도 2A). 이러한 현상을 유도만능줄기세포에서 특이적 활성을 보이는 알칼린포스타제 염색법을 통하여 더 확인할 수 있었다(도 2B).After transfection of reprogramming factors into cells cultured on
<< 실시예Example 2> 방사선 조사 후 장기배양된 세포로부터 리프로그램된 세포들의 만능줄기세포 특성 분석 2> Characterization of pluripotent stem cells from reprogrammed cells from long-term cultured cells after irradiation
리프로그램된 세포에서 만능줄기세포의 형태학적 특징, 알칼린포스타제 활성도 및 만능줄기세포 마커 단백질들 (Nanog, Oct4 및 Sox2) 발현을 면역염색을 통해 확인하였다(도 3).The morphological characteristics of the pluripotent stem cells, alkaline phosphatase activity and the expression of the pluripotent stem cell marker proteins (Nanog, Oct4 and Sox2) in the reprogrammed cells were confirmed by immunostaining (Fig. 3).
리프로그램된 세포에서 만능줄기세포 마커 유전자들 (Nanog, Oct4 및 Sox2) 발현을 real-time PCR을 통해 확인하였다(도 4).The expression of pluripotent stem cell marker genes (Nanog, Oct4 and Sox2) in reprogrammed cells was confirmed by real-time PCR (Fig. 4).
결론적으로, 방사선 전조사는 (pre-irradiation) 유도만능줄기세포 리프로그래밍을 저해하지만, 방사선 조사 후 (post-irradiation)은 유도만능줄기세포 리프로그래밍 촉진을 확인할 수 있었다. 또한, 만능줄기세포 특성 분석을 통해 방사선 조사 후에 의해 고효율로 리프로그램된 세포들이 유도만능줄기세포임을 증명하였다. 따라서, 이러한 결과는 방사선 조사 후 활성화된 인자들이 유도만능줄기세포 리프로그래밍 증진에 관여함을 시사하고 있다.In conclusion, pre-irradiation inhibits inducible pluripotent stem cell reprogramming, but post-irradiation promotes inducible pluripotent stem cell reprogramming. In addition, by analyzing the characteristics of the pluripotent stem cells, it was proved that highly reprogrammed cells were induced pluripotent stem cells after irradiation. Thus, these results suggest that activated factors after irradiation may be involved in promoting induced pluripotent stem cell reprogramming.
상기와 같은 결과는 유도만능줄기세포 리프로그래밍 효율증진 인자 탐색 및 기전 연구에 활용 될수 있을 것으로 기대된다.These results are expected to be useful for the investigation of the inducible pluripotent stem cell reprogramming efficiency factor and its mechanism.
Claims (4)
(2) 상기 방사선이 조사된 섬유아세포를 10 내지 25일 배양하는 단계; 및
(3) 상기 배양된 섬유아세포를 유도만능줄기세포로 리프로그래밍시키는 단계를 포함하는 방사선 조사 후 장기 세포배양에 의한 유도만능줄기세포 리프로그래밍 효율 증진 방법.(1) irradiating fibroblasts with radiation;
(2) culturing the irradiated fibroblasts for 10 to 25 days; And
(3) Reprogramming the cultured fibroblasts to inducible pluripotent stem cells. The method of promoting induced pluripotent stem cell reprogramming by long-term cell culture after irradiation.
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