본 연구는 단위발생유래 생쥐 배아줄기세포(P-mES)지가 체외수정유래 생쥐 배아줄기세포 (mES)와 마찬가지로 기능성 심근세포로 체외 분화되는지를 조사하였다. 각 세포주 P-mES04와 MES03를 4일간 부유 배양하여 배아체 (EB)를 형성한 다음 4일간 DMSO를 추가적으로 처리한 뒤 젤라틴이 코팅된 배양접시에 부착시켰다(4-/4+). P-mES04와 mES03으로부터 수축성 심근세포 생성 여부를 30일간 관찰한 결과, 각각 13일(69.83%)과 22일 (61.3%)에 누적 형성율이 가장 높았다. 면역 세포화학염색 결과, 수축성을 나타내는 P-mES04 세포는 수축성 mES03 세포에서와 같이 근육 특이적인 anti-sarcomeric a-actinin 항체와 심근 특이적인 anti-cardiac troponin I 항체에 염색되는 것을 확인하였다. 또한 RT-PCR 결과, 수축성을 나타내는 P-mES04 세포는 심근특이적인 L-type calcium channel, a1C, cardiac myosin heavy chain a, cardiac muscle heavy polypeptide 7β, GATA binding protein 4와 atrial natriuretic factor는 발현하나, 골격근 특이적인 L-type calcium channel, a1S는 발현하지 않아 웅성 성체의 심장세포와 유사한 양상을 보였다. 본 연구의 결과는 단위발생 유래 생쥐 배아 줄기세포를 배아줄기세포의 연구의 대체제로 이용할 수 있음을 보여준다.

Human embryonic stem (hES) cells can be induced to differentiate into tyrosine hydroxylase expressing (TH+) cells that may serve as an alternative for cell replacement therapy for Parkinson's disease (PD). To examine in vitro differentiation of hES (MB03, registered in NIH) cells into TH+ cells, hES cells were induced to differentiate according to the 4-/4+ protocol using retinoic acid (RA), ascorbic acid (AA), and/or lithium chloride (LiCl) followed by culture in N2 medium for 14 days, during which time the differentiation occurs. Immunocytochemical stainings of the cells revealed that approximately 21.1% of cells treated with RA plus AA expressed TH protein that is higher than the ratio of TH+ cells seen in any other treatment groups (RA, RA+LiCl or RA+AA+LiCl). In order to see the differentiation pattern in vivo and the ability of in vitro differentiation-induced cells in easing symptomatic motor function of PD animal model, cells (2 10 cells/2) undergone 4-/4+ protocol using RA plus AA without any further treatment were transplanted into unilateral striatum of MPTP-lesioned PD animal model (C57BL/6). Following the surgery, motor behavior of the animals was examined by measuring the retention time on an accelerating rotar-rod far next 10 weeks. No significant differences in retention time of the animals were noticed until 2 weeks post-graft; however, it increased markedly at 6 weeks and 10 weeks time point after the surgery. Immunohistochemical studies confirmed that a reasonable number of TH+ cells were found at the graft site as well as other remote sites, showing the migrating nature of embryonic stem cells. These results suggest that in viかo differentiated hES cells relieve symptomatic motor behavior of PD animal model and should be considered as a promising alternative for the treatment of PD.