Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal and differentiation into a variety of cell types. They represent an attractive source of cells for gene and cell therapy. The purpose of this study is to direct the specific expression of the DsRed reporter gene in Sca-1+ BMMSCs differentiated into a cardiomyogenic lineage. We constructed the prMLC-2v-DsRed vector expressing DsRed under the control of the 309 tp fragment of the rat MLC-2v 5'-flanking region. The specific expression of the DsRed reporter gene under the transcriptional control of the 309 bp fragment of the rat MLC-2v promoter was tested in 5-azacytidine healed-Sca-1+ BMMSCs over 2 weeks after the prMLC-2v-DsRed transfection. The prMLC-2v-DsRed was specifically expressed in the Sca-1+ BMMSCs with cardiomyogenic lineage differentiation and it demonstrates that the 309 bp sequences of the rat MLC-2v 5'-flanking region is sufficient to confer cardiac specific expression on a DsRed reporter gene. The cardiac-specific promoter-driven reporter vector provides an important tool for the study of stem cell differentiation and cell replacement therapy in ischemic cardiomyopathy.

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.

Pluripotent embryonic stem (ES) cells differentiate spontaneously into beating cardiomyocytes via embryo-like aggregates. We describe the use of mouse embryonic stem (mES03) cells as a reproducible differentiation system for cardiomyocyte. To induce cardiomyocytic differentiation, mES03 cells were dissociated and allowed to aggregate (EB formation) at the presence of 0 75% dimethyl sulfoxide (DMSO) for 4 days and then another 4 days without DMSO (4+/4-). Thus treated EBs were plated onto gelatin-coated dish for differentiation. Spontaneously contracting colonies which appeared in approximately 4-5 days upon differentiation. Expression of cardiac-specific genes were determined by RT-PCR. Rebust expression of myosin light chain (MLC-2V), cardiac myosin heavy chain , cardiac muscle heavy polypeptide 7 -MHC), cardiac transcription factor GATA4 and skeletal muscle-specific -subunit of the L-type calcium channel () were detected as early as 8 days after EB formation, but message of cardiac muscle-specific -subunit of the L-type calcium channel (CaCh) were revealed at a low level. Strikingly, the expression of atrial natriuretic factor (ANF) was not detected. When spontaneous contracting cell masses were examined their electrophysiological features by patch-clamp technique, it showed ventricle-like action potential 17 days after the EB formation. This study indicates that mES03 cell-derived cardiomyocytes displayed biochemical and electrophysiological properties of cardiomyocytes and DMSO enhanced development of cardiomyocytes in 4+/4- method.