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.

Mammalian Emx2. a homeobox tra nscripti on factor‘ is continuoll s ly expressecl in aclll lt neural s tem cell s of the b.ippo campal c1enclate gyrus (HDG) a f'ter blrth 1'0 c1ate‘ roles 01' Emx2 a ncl its llnderlying rnecha ni s rn in r eg비 atin g acl lllt neuro - genesis from neural stem cell aft er bi rth is still obscure. 1'he present experiment is aimed to elucidate role 01' Emx2 in regulating adll lt neurogenesis from neural s tem cell of HDG using nestin-Emx2 transgenic mouse (N-E2 1'G) and heterozygous Emx2 KO mouse (1-l-E2 KO) . HDG g ranlllar cell layer where new born neurons proclllced from adult neural stem cell migrate. is thin with low cell c1ens ity in N-E2 1'G but tbick with high cell density in H-E2 KO, compared to wild type mice (\\끼') . Number of DCX , a new born nellron marker. -positive cells is less in N-E2 1'G but more in l-l-E2 KO. comparecl to W1'. Ki67 (whole cell cycle marker) 01' BrclU (S-phase marker) 一positive cells is less in N-E2 1'G bllt morc in l-l-E2 KO and BrdU-positive cells/ Ki 67ratio is higher in N-E2 1'G but lower in H-E2 KO. NeuN (a mature n e llro삐 marker) a ncl BrdU-dollble positive cells is lUore in N- E2 TG bllt GFAP (a glial cell marker) ancl BrdU- dollble positive cells is more in ]-]- E2 KO. compa recl to WT 4wks after BrclU is aclmin istratecl one ti me per c1ay for 5days‘ Migrating c1egree of BrdU-positive cells is lower in N-E2 TG but higher in ]-]-E2 KO 4wks after BrclU is administratecl one t ime per day for 5days. Active casepase 3-positive cells is more in ]-]DG 01' the N-E2 TG but no changes in ]-]-E2 KO. 4 wks after CAG- GFP- PRE vector was inj ected in hippocampus. GFP-positive new born n e urons from aclult neural stem cell have less c1endritic branches in N-E2 1'G but more c1endritic branches in H-E2 KO‘ comparecl to the WT From these results. Emx2 transcription factor inhibits adult neurogenesis f'rom nellral stem cell of HDG throllgh reducing neural stem cell proliferation. new born cell survival. ce ll migration. ancl matllrat ion

For many years, experience has been accumulated on embryo and gamete manipulation in mammals, The present work is an introduction of these techniques and their possible application in human embryology in s pecific cases, Mammalian c1on ing has been studied by many groups, but the success rate is sti ll low‘ Removal of maternal chromosomes from unfertil ized oocytes and injection of donor cells into enucleated oocytes are the most important factors for the improvement of cloning effi cien cy, Here, we introduce a novel one-step rnicromanipulation (OSM) system and laser-assisted zona pellucida piel'cing technique (LAZP) , 1n genera l, somatic cell nuclear transfer (SCNT) is completed by many processes including enucleation and donor cell fusion , Howevel', OSM is a simple method because donor cell is directly injected into ooplasm without fusion pl'ocess, 1n addition, chromosomal enucleation and donor cell inj ec tion are perfOl‘med simultaneously in OSM, While OSM was a pplied to porcine SCNT, LAZP was a pplied to murine SCNT, This rninirni zed the use of piezo-dri ven micromanipul ator (P1EZO) , I'educing chances 0 1' problems caused by P1EZO pulses, LAZP reduced time that took to pierce zona pellucida in removal of nucleus fl'om oocyte and somatic cell injection, which might have taken longer time with P1EZO, The simple , new OSM and LAZP system may help to enable large scale cloning by reduction of procedural steps, Pa l'thenogenesis de scribes the growth and development of an embryo without fertilization by a male Parthenogenetic ES cell s (PESCs) can be a useful cell source for tissue I'epail‘ and I'egeneration , Moreover , the defects in full-term developrnent of this PESCs enable researc hers to avoid the ethical concern , Here, the author showed that PESCs can differentiate into osteogenic lineage, The PESCs were induced osteogenic dlfferentlatlon The osteoblas t-specific gene expression such as osteocalcine, osteopontine, osteonectin, bone-sialo protein‘ coll agen type-l and alka line phos phatase showed osteogenic potential of differentiated PESCs, The author also focused on the neuronal induction of murine PESCs by simplified neurona l induction system to generate doparninergic (DA) neurons , As a result , PESCs were differentiated into nestin and Tuj-l positive cell s successfully, a lthough t he generation of DA neuron was Illruted For murine embryo cul ture, novel oil-free microtube cul tu re system was applied , This new culture system provides oil-free cu ltu re condi t ions and is easy to handle It was also associated with faster development and mOl'e t l'ophectodel'mal cells , which will enhance the development of murine embl'Yos to fur t hel' stages ,

본 연구는 소 배반포의 내부 세포괴로부터 다능성(pluripotency)을 지닌 배아 줄기 세포(embryonic stem cell) 또는 그 유사 세포를 분리 및 배양함으로써 줄기 세포 관련 분야의 기반 기술을 확립하고자 하였다. 소 체외수정란을 10～12일간 체외배양하여 생산된 부화 배반포를 세포분열이 불활성화된 생쥐 태아 섬유아 세포(mouse embryonic fibroblast, MEF) 위에서 배양하여 콜로니 형성을 유도하였으며, 이들로부터 내부 세포괴 유래의 형태를 지닌 것만을 광학현미경 하에서 물리적으로 분리하여 약 5～7일 간격으로 계대배양을 실시하였다. 이러한 방법을 통하여 배아 줄기 유사 세포의 특성을 40계대 이상 유지하는 2개의 세포주를 확립하였다. 각각의 세포주들은 높은 alkaline phosphatase(AP) 활성을 지니고 있었으며, 형광 면역 염색법과 PCR 기법을 사용하여 Oct-4, Nanog, STAT3, SSEA3 및 SSEA4의 발현을 관찰할 수 있었다. 이러한 결과를 종합하여 볼 때 ,본 연구에서는 소 배반포로부터 배아 줄기 세포주를 확립하는 제반 기술이 확립되었다고 판단되며, 향후 관련 분야 연구에 활용될 수 있을 것으로 기대된다.