일반적으로 세포·조직 및 장기이식 성공 예측은 수여자와 공여자간의 백혈구항원 일치도이고, 불일치 시 심각한 거부 반응을 유발함으로 세포치료제로 사용할 때 우선적으로 백혈구 항원일치도가 고려된다. 그러나 중간엽줄기세포(Mesenchymal Stem Cells, MSCs)는 다른 체세포와 비교하여 상대적으로 낮은 MHC I 항원발현과, 극히 낮은 MHC II 항원을 가지고 있으므로 동종세포치료제로서 주목을 받고 있다. 따라서 본 연구에서는 개 모델에서 MSCs 의 동종세포치료제로서 효능을 예측하기 위해 선행연구로 백혈구 항원(Dog Leukocyte Antigen, DLA)형 및 가계도내 일치도와 유전적다형성(Polymorphism) 을 분석하였다. DLA 분석을 위해 한가계도의 비글(Covance Beagles) 4 두(모견 1 두, 자견 3 두)로 부터 전혈을 채취하고, 밀도구배를 이용하여 백혈구만을 분리 후 DNA들을 각각 추출하였다. DLA 분석은 ClassII 유전자(DLA-DQA, DLA-DQB, DLA-DRB)에서 엑손 2 영역(약 300bp)을 증폭하고 Direct Sequencing 을 통해 밝혀진 염기서열을 NCBI Blast 와 IPD(Immuno Polymorphism Database)를 기반으로 하여 Universal nomenclature 에 따라 유전자형을 판독 하였다. 그 결과 DLA-DQA(022:01/022:01)와 DLA-DQB(107:01/102:01)는 4마리 모두 유전자형이 동일하였으나, DLA-DQB 는 각각 046:01/022:02, 03701/022:02, 00201/022:02, 03701/022:02 로 차이를 보였다. 이 결과를 통해 모견과 자견이 공통적으로 가지는 일배체형(Haplotype)은 DLA–DQA*022:01, DLA-DQB*022:02, DLA-DRB*102:01 이었음을 확인할 수 있었다. 그리고 일부 유전자의 염기서열에서 99% 유사도를 보이는 후보군들이 4 개씩 검색되었는데 이는 단일염기다형성(SNP)에 기인한 유전적다형성(Polymorphism)이 매우 높다는 선행보고들과 유사한 결과를 보였다. 본 실험결과는 향후 DLA 의 일치군과 비 일치군의 개중간엽줄기세포와 말초혈액단핵구세포(PBMC)들의 공배양을 통해 동종세포치료제 연구에 사용될 예정이다. * 본 성과물은 농촌진흥청 반려동물 연구사업(세부과제명 : 반려견에서 DLA 일치하는 줄기세포의 체외 치료능 평가, 세부과제 번호 : PJ013957022018)의 지원에 의해 이루어짐.

Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of β-cells, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from α-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3∼4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in β-cell replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

Mesenchymal stem cell (MSC) based cell therapy has emerged as a promising therapeutic approach for treatment of several degenerative, infectious and non-infectious diseases. Numerous studies have demonstrated the remarkable immunosuppressive and antibacterial effects of MSCs both in vitro and in vivo, in animal models and in humans. However, the antibacterial effects of MSCs rely heavily on their paracrine factors rather than direct cell-to-cell contact and the effect is specific to disease and site of infection or injury. Furthermore, recent studies have demonstrated the double-edged sword effect of MSCs in bacterial infectious diseases. Despite their inherent potential for repair of damaged tissues, immunosuppression, and alleviation of various autoimmune as well as infectious diseases, MSCs also play a critical role in promoting persistent bacterial infection and disease progression. Therapeutic administration of MSCs successfully inhibited the bacterial growth and enhances survival by improved clearance of pathogenic bacteria in sepsis and pneumonic conditions. However, due to their abnormal transformation, they assist in long lasting survival and persistent infection of Mycobacterium tuberculosis (M. tuberculosis) and may also be responsible for progression of gastric cancer. This review focuses on recent advances that have broadened our understanding of MSC based therapy for bacterial diseases and provides new insight into the possible therapeutic targets of fatal bacterial diseases.

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal α -1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from α-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% CO2 incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers (CD45－, 29+, 90+ and 105+) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited CD45－, 29+, 90+ and 105+ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at 1×103 cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs (15.0 ± 0.4 μm) had a little larger cell size than Wild BM-MSCs (13.5 ± 0.3 μm). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

Adult stem cell transplantation has been increased every year, because of the lack of organ donors for regenerative medicine. Therefore, development of reliable and safety cryopreservation and bio-baking method for stem cell therapy is urgently needed. The present study investigated safety of dimethyl sulfoxide (DMSO) such as common cryoprotectant on porcine bone marrow derived mesenchymal stem cells (pBM-MSCs) by evaluating the activation of Caspase-3 and -7, apoptosis related important signal pathway. pBM-MSCs used for the present study were isolated density gradient method by Ficoll-Paque Plus and cultured in A-DMEM supplemented 10% FBS at in 5% incubator. pBM-MSCs were cryopreserved in A-DMEM supplemented either with 5%, 10% or 20% DMSO by cooling rate at /min in a Kryo 360 (planner 300, Middlesex, UK) and kept into . Survival rate of cells after thawing did not differ between 5% and 10% DMSO but was lowest in 20% DMSO by 0.4% trypan blue exclusion. Activation of Caspase-3 and -7 by Vybrant FAM Caspase-3 and -7 Assay Assay Kit (Molecular probes, Inc.OR, USA) was analyzed with a flow cytometer. Both of cryopreserved and control groups (fresh pBM-MSCs) were observed after the activation of Caspase-3 and -7. The activation did not differ between 5% and 10% DMSO, but was observed highest in 20% DMSO. Therefore 5% DMSO can be possibly used for cell cryopreservation instead of 10% DMSO.