Immunosuppression-enhancing effect of the administration of allogeneic canine adipose-derived mesenchymal stem cells (cA-MSCs) compared with autologous cA-MSCs in vitro

위하연 대한수의학회 2021 Journal of Veterinary Science Vol.22 No.5

Background: Recently, mesenchymal stem cells therapy has been performed in dogs, although the outcome is not always favorable. Objectives: To investigate the therapeutic efficacy of mesenchymal stem cells (MSCs) using dog leukocyte antigen (DLA) matching between the donor and recipient in vitro. Methods: Canine adipose-derived MSCs (cA-MSCs) isolated from the subcutaneous tissue of Dog 1 underwent characterization. For major DLA genotyping (DQA1, DQB1, and DRB1), peripheral blood mononuclear cells (PBMCs) from two dogs (Dogs 1 and 2) were analyzed by direct sequencing of polymerase chain reaction (PCR) products. The cA-MSCs were co-cultured at a 1:10 ratio with activated PBMCs (DLA matching or mismatching) for 3 days and analyzed for immunosuppressive (IDO, PTGS2, and PTGES), inflammatory (IL6 and IL10), and apoptotic genes (CASP8, BAX, TP53, and BCL2) by quantitative real-time reverse transcriptase-PCR. Results: cA-MSCs were expressed cell surface markers such as CD90+/44+/29+/45− and differentiated into osteocytes, chondrocytes, and adipocytes in vitro. According to the Immuno Polymorphism Database, DLA genotyping comparisons of Dogs 1 and 2 revealed complete differences in genes DQA1, DQB1, and DRB1. In the co-culturing of cA-MSCs and PBMCs, DLA mismatch between the two cell types induced a significant increase in the expression of immunosuppressive (IDO/PTGS2) and apoptotic (CASP8/BAX) genes. Conclusions: The administration of cA-MSCs matching the recipient DLA type can alleviate the need to regulate excessive immunosuppressive responses associated with genes, such as IDO and PTGES. Furthermore, easy and reliable DLA genotyping technology is required because of the high degree of genetic polymorphisms of DQA1, DQB1, and DRB1 and the low readability of DLA 88.

[청년의 활동] 행복을 나누는 음악 바투카다를 통해 연대를 배우다

위하연 (주)유레카엠앤비 2021 유레카 Vol.- No.452

개 중간엽줄기세포(Mesenchymal Stem Cells, MSCs)의 면역관용능 평가를 위한 백혈구 항원타입 동정

위하연,김영임,노진구,이란,우제석,이승훈,허태영,옥선아 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

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.

[서술형 배경지식] 인포그래픽이 지닌 몇 가지 장점

위하연 (주)유레카엠앤비 2021 유레카 Vol.- No.452

Effect of Wnt signaling pathway activation on the efficient generation of bovine intestinal organoids

박강원,양현,위하연,옥선아,이풍연,황인설,이보람 사단법인 한국동물생명공학회 2022 한국동물생명공학회지 Vol.37 No.2

Recent progress has been made to establish intestinal organoids for an in vitro model as a potential alternative to an in vivo system in animals. We previously reported a reliable method for the isolation of intestinal crypts from the small intestine and robust three-dimensional (3D) expansion of intestinal organoids (basal-out) in adult bovines. The present study aimed to establish next-generation intestinal organoids for practical applications in disease modeling-based host-pathogen interactions and feed efficiency measurements. In this study, we developed a rapid and convenient method for the efficient generation of intestinal organoids through the modulation of the Wnt signaling pathway and continuous apical-out intestinal organoids. Remarkably, the intestinal epithelium only takes 3-4 days to undergo CHIR (1 µM) treatment as a Wnt activator, which is much shorter than that required for spontaneous differentiation (7 days). Subsequently, we successfully established an apical-out bovine intestinal organoid culture system through suspension culture without Matrigel matrix, indicating an apical-out membrane on the surface. Collectively, these results demonstrate the efficient generation and next-generation of bovine intestinal organoids and will facilitate their potential use for various purposes, such as disease modeling, in the field of animal biotechnology.

개 parthenote in vitro culture시 EDTA 첨가에 의한 발달율 향상

정해윤,노진구,이휘철,위하연,옥선아,우제석,허태영,임기순,김종국,이승훈,Jeong, Haeyun,Zhao, Minghui,No, Jin-Gu,Ullah, Imran,Lee, Whi-Cheul,Wi, Hayeon,Ock, Sun A,Hur, Tai-young,Woo, Jae-Seok,Im, Gi-sun,Kim, Jong-Gug,Lee, Seunghoon 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.3

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

Induction of the differentiation of porcine bone marrow mesenchymal stem cells into premature hepatocyte-like cells in an indirect coculture system with primary hepatocytes

Ullah Imran,서강민,위하연,김영민,이승훈,옥선아 한국통합생물학회 2020 Animal cells and systems Vol.24 No.5

Liver transplantation is currently the only option for patients with end-stage liver disease. Thus, other alternate therapeutic strategies are needed. Bone marrow mesenchymal stem cells (BMMSCs) are nonhematopoietic cells present in the bone marrow stroma that serve as precursors cells for various other cells. In this study, we evaluated the differentiation of porcine BM-MSCs into hepatocyte-like cells using three types of culture systems: hepatic induction medium (HIM), HIM/primary hepatocyte culture supernatant (HCS; 1:1 ratio), and a hepatocyte coculture system (HCCS; primary hepatocytes in the upper chamber, and BM-MSCs in the lower chamber). Primary hepatocytes were isolated from anesthetized healthy 1-month-old pigs by enzymatic digestion. Hepatic-specific marker expression (albumin [ALB], transferrin [TF], α-fetoprotein [AFP]), glycogen storage, low-density lipoprotein, and indocyanine green uptake were evaluated. Upregulation of hepatic-specific markers (ALB, TF, and AFP) was observed by real-time polymerase chain reaction in the HCCS group. Periodic acid-Schiff staining revealed enhanced glycogen storage in hepatocyte-like cells from the HCCS group compared with that from the HIM/HCS group. Furthermore, hepatocyte like-cells in the HCCS group showed improved LDL and ICG uptake than those in the other groups. Overall, our current study revealed that indirect coculture of primary hepatocytes and BM-MSCs enhanced the differentiation efficacy of BM-MSCs into hepatocyte-like cells by unknown useful soluble factors, including paracrine factors.

혈청이 마우스 근육세포 증식에 미치는 효과

이란,서강민,김영임,임란,위하연,이풍연,옥선아 경상대학교 농업생명과학연구원 2020 농업생명과학연구 Vol.54 No.6

이 연구의 목적은 근육 세포의 증식 배양을 위해 필요한 핵심 인자인 혈청이 혈청 대체물의 첨가에 의해 대체될 수 있는지를 분자 생물학적 측면에서 검증하는 것이다. 혈청 대체물이 첨가된 low serum (5% FBS) 기반의 promo cell 배지는 성장 배양액으로 사용되었고, 마우스 하지 골격근의 근육세포는 pre-plating (pp) 방법에 의해 분리되었다. Pre-plating 4의 세포는 작고 둥근 형태의 굴절성을 가진 Myoblast/satellite like cells의 형태가 관찰되었으며, 근육 줄기세포 전사인자들(Pax3/7, Myf5, Myod1)과 골격근 발달 전사인자(Myog)의 발현량이 섬유아세포와 비교하여 높게 나타났다. 따라서 그들을 Myoblast derived cells로 명명하고, 기본 세포주로 사용하였다. 분리된 MDCs는 5%, 10% 혹은 20% 혈청이 첨가된 배양액에서 2주간 배양되었다. 배양 6일째부터 대조군(5% 혈청)과 비교하여 20% 혈청은 세포 수가 증가하였으며, 양적 혈청 농도 의존성이 확인되었다. 증식 및 세포사멸 관련 유전자들은 대조군과 비교하였다. 배양 1주 차에 20% FBS군은 세포증식 촉진 유전자인 Myc 발현이 증가한 반면 pro-apoptosis 유전자인 Bax의 발현량이 감소했고, 2주 차에는 세포주기정지 인자인Cdkn1a의 감소와 Myc의 지속적인 증가와 Bax/Bcl의 감소가 나타났다. 각기 다른 FBS 처리농도에서 배양된 Myoblast derived cells을 동일한 Myotube유도 배양액에서 2주간 분화하였다. 대조군과 비교하여 20% FBS군은 Myod1, Myog, Myf6, Myh1의 유의적 증가가 1주부터 확인되었다. 결론적으로 혈청 대체 물질은 근육세포 배양액에서 혈청의 효과를 완벽히 모사할 수 없음이 증명되었고, 따라서 체외에서 상업적 목적의 근육 세포 대량 증식 및 분화를 위해서는 적절한 혈청 대체물 개발이 선행돼야 할 것으로 사료된다. This study was performed to examine whether serum used for growth of skeletal muscle cells in vitro can be replaced by serum substitutes or not. Briefly, It was used commercial Promo cell growth medium with serum replacements (5% FBS) as a basal growth medium (GM). Mouse skeletal muscle cells were isolated from hind limb by Pre-plating (pp) technology. The isolated cells at pp 4 stage showed small and round morphology with refractive looks like myoblast/satellite like cells, and showed increase of Pax3/7, Myf5, Myod1 and Myog expression compared to fibroblasts. The cells were then named as myoblast derived cells (MDCs), and were used for the further experiments. MDCs were cultured into GM supplemented with 5, 10 or 20% FBS for 2 weeks. The total cells number were calculated after every 2 days using a hemocytometer. The number of cells increased significantly in 20% FBS cultured cells as compared to control (≥6 days) and was positively correlated with FBS concentration. Proliferation and apoptosis related genes were analyzed by real time PCR. It was shown the upregulation of Myc expression and downregulation of Bax by 20% FBS treatment at 1 week. Furthermore, there was reduction of Cdkn1a/ Bax/Bcl2 and increase of Myc expression at 2 weeks in 20% FBS cultured cells compared to control. The MDCs were differentiated with Promocell differentiation media (serum free) for 2 weeks, where 20% FBS compared to control showed significantly increased in expression of Myod1/Myog/Myf6/ Myh1 from 1 to 2 weeks. In conclusion, serum substitutes could not fully mimic the properties of the serum in the muscle cell culture. Therefore, for mass production of muscle cells for commercial purposes, it is necessary to develop the suitable serum to overcome cost and the ethical implications of extracting FBS.

Establishment of intestinal organoids from small intestine of growing cattle (12 months old)

박강원,Hyeon Yang,Min Gook Lee,옥선아,위하연,이풍연,In-Sul Hwang,유재규,Choon Keun Park,Bo Ram Lee 한국축산학회 2022 한국축산학회지 Vol.64 No.6

Recently, we reported the robust in vitro three-dimensional (3D) expansion of intestinal organoids derived from adult bovine (> 24 months) samples. The present study aimed to establish an in vitro 3D system for the cultivation of intestinal organoids derived from growing cattle (12 months old) for practical use as a potential alternative to in vivo systems for various purposes. However, very few studies on the functional characterization and 3D expansion of adult stem cells from livestock species compared to those from other species are available. In this study, intestinal crypts, including intestinal stem cells, from the small intestines (ileum and jejunum) of growing cattle were isolated and long-term 3D cultures were successfully established using a scaffold-based method. Furthermore, we generated an apical-out intestinal organoid derived from growing cattle. Interestingly, intestinal organoids derived from the ileum, but not the jejunum, could be expanded without losing the ability to recapitulate crypts, and these organoids specifically expressed several specific markers of intestinal stem cells and the intestinal epithelium. Furthermore, these organoids exhibited key functionality with regard to high permeability for compounds up to 4 kDa in size (e.g., fluorescein isothiocyanate [FITC]-dextran), indicating that apical-out intestinal organoids are better than other models. Collectively, these results indicate the establishment of growing cattle-derived intestinal organoids and subsequent generation of apical-out intestinal organoids. These organoids may be valuable tools and potential alternatives to in vivo systems for examining host-pathogen interactions involving epithelial cells, such as enteric virus infection and nutrient absorption, and may be used for various purposes.