Nonmuscle myosin II가 인간 배아줄기세포 분화에 미치는 영향에 관한 연구

Abstract

인간 배아줄기세포는 수정란의 내부 세포 덩어리에서 유래한 세포주로서, 3배엽(내배엽, 중배엽, 외배엽)으로 분화 할 수 있는 전분화능(pluripotency)을 특징으로 한다. 이러한 인간 배아줄기세포는 배아에서 유래한 전분화능 세포의 기능을 반영하기 때문에 배아의 증식과 분화에 관련한 연구 재료로 활용될 수 있다. 최근 세포 골격 조절 인자인 Rho-associated kinase(ROCK)와 nonmuscle myosin II(NMII)의 발현을 억제함으로써 인간 배아줄기세포의 생존율과 미분화 유지를 효과적으로 증진할 수 있다는 보고들이 있다. 본 연구에서는 세포 골격을 조절하는 NMII의 isoform 인 myosin, heavy chain 9, non-muscle (MYH9)과 myosin, heavy chain 10, non-muscle (MYH10)이 인간 배아줄기세포의 분화에 미치는 영향에 대하여 연구하였다. 인간 배아줄기세포의 분화를 유도한 배상체를 실험 재료로 사용하였으며, 배아줄기세포가 분화할수록 MYH9과 MYH10 발현이 증가하는 현상을 유전자와 단백질 발현 수준에서 확인하였다. MYH9과 MYH10 발현 양상을 조사하기 위하여 면역화학염색을 수행하였다. 미분화 배아줄기세포에서는 발현을 확인할 수 없었으나, 분화한 배아줄기세포에서는 세포 전반에서 발현하는 것을 확인하였다. 이렇게 분화에 따라 NMII 발현이 증가하는 현상이 배아줄기세포의 분화에 어떤 영향을 미치는지 조사하기 위하여 NMII의 기능을 억제하는 약물인 blebbistatin을 처리한 후 분화에 미치는 영향을 조사하였다. 약물 처리 농도는 줄기세포, 암 세포주 그리고 배아에서 약물의 효과를 보고한 논문들을 조사하여 10uM, 50uM, 100uM 로 다양하게 처리하였으며, blebbistatin 처리에 따라 NMII 발현이 감소하는 것을 유전자와 단백질 발현 수준에서 확인하여 약물 처리 효과를 검증 하였다. 약물 처리 시간과 시기에 따른 배양조건을 3가지로 하였으며, 각 조건에 따른 배상체의 형태와 유전자의 변화를 관찰하였다. 실험결과 약물 농도가 높을수록, 약물 처리 시간이 길수록 배상체 형성을 저해하며, 내배엽과 중배엽 분화가 억제되는 것을 관찰 할 수 있었는데, 이러한 현상은 배상체 형성과 동시에 약물을 처리하였을 때는 관찰할 수 있었지만 배상체 형성이 진행된 후 약물 처리를 하면 관찰할 수 없었다. 결론적으로 본 연구에서는 NMII 발현저하가 배상체 형성을 저해하여 배아줄기세포의 초기 분화에 영향을 미치는 것으로 조사되었고, 상세한 기전 분석연구가 추가적으로 필요할 것으로 사료된다. | Human embryonic stem cells(hESCs) derived from the inner cell mass(ICM) of human blastocysts are capable of unlimited growth and differentiating into all three germ layers. Because hESCs mirror the pluripotency functions for studying early human development, they can be used as model systems to understand the mechanisms underlying basic cytoskeleton in early mammalian embryos. Especially recent reports revealed that the use of the ROCK inhibitor Y-27632 and nonmuscle myosin II (NMII) inhibitor blebbistatin increases cell survival and cloning efficiency of hESCs. These accumulated knowledge of cytoskeleton prompted us to investigate whether NMII plays important roles in differentiation of hESCs. General differentiation method into three germ layers has been used hESC-derived embryoid bodies (hEBs) formation. We show that a increase of NMII isoform, MYH9 and MYH10, in spontaneously differentiating hESCs by genes and proteins levels. We next examined the expression pattern of MYH9 and MYH10 within hESCs and hEBs using immunocytochemistry. MYH9 and MYH10 was highly expressed and broadly distributed in the hEBs cells, but it was not detectable in hESCs. To determine whether down-regulation of MYH9 and MYH10 affected the differentiation of hESCs, we knocked down these proteins in hEBs by a specific inhibitor of nonmuscle myosin II, blebbistatin. Experimental design of blebbistatin treatment in hEBs was 3 groups. Blebbistatin added in hEB culture medium at 0 day for 3 days (group1). In group 2 and 3, blebbistatin treated only 7hrs at 0 day hEBs and 3 days hEBs, then cultured for 3 more days. In each group, medium contained blebbistatin at 3 different concentrations; 10μM, 50μM and 100μM. To determine effect of blebbistatin treatment in hEBs development, we examined the MYH10 expression levels in blebbistatin treated hEBs.. After blebbistatin treatment, hEBs formation of each group was observed by phase-contrast microscope. We concluded that NMII inhibition affects the normal hEBs formation by exposed time, concentration and immediate treatment of blebbistatin manner. And we showed that differentiation marker gene expression level was decreased by blebbistatin treatment, especially endoderm and mesoderm lineage. These results suggest that NMII inhibitor blebbistatin may affect normal hEBs formation and NMII may influence on early hESCs differentiation.; Human embryonic stem cells(hESCs) derived from the inner cell mass(ICM) of human blastocysts are capable of unlimited growth and differentiating into all three germ layers. Because hESCs mirror the pluripotency functions for studying early human development, they can be used as model systems to understand the mechanisms underlying basic cytoskeleton in early mammalian embryos. Especially recent reports revealed that the use of the ROCK inhibitor Y-27632 and nonmuscle myosin II (NMII) inhibitor blebbistatin increases cell survival and cloning efficiency of hESCs. These accumulated knowledge of cytoskeleton prompted us to investigate whether NMII plays important roles in differentiation of hESCs. General differentiation method into three germ layers has been used hESC-derived embryoid bodies (hEBs) formation. We show that a increase of NMII isoform, MYH9 and MYH10, in spontaneously differentiating hESCs by genes and proteins levels. We next examined the expression pattern of MYH9 and MYH10 within hESCs and hEBs using immunocytochemistry. MYH9 and MYH10 was highly expressed and broadly distributed in the hEBs cells, but it was not detectable in hESCs. To determine whether down-regulation of MYH9 and MYH10 affected the differentiation of hESCs, we knocked down these proteins in hEBs by a specific inhibitor of nonmuscle myosin II, blebbistatin. Experimental design of blebbistatin treatment in hEBs was 3 groups. Blebbistatin added in hEB culture medium at 0 day for 3 days (group1). In group 2 and 3, blebbistatin treated only 7hrs at 0 day hEBs and 3 days hEBs, then cultured for 3 more days. In each group, medium contained blebbistatin at 3 different concentrations