본 연구에서는 보령댐 유역(163.6 km2)을 대상으로 SWAT (Soil and Water Assessment Tool) 모델, GCM (General Circulation Model) 기 후변화 시나리오와 다중회귀분석으로 산정한 미래 방류량을 활용하여 극한 기후변화 사상이 반영된 보령댐의 물부족을 평가하였다. 유역의 물수지 분석을 위해 보령댐 유역을 대상으로 기상자료, 보령댐 운영자료를 수집하였으며, SWAT 모형의 신뢰성 있는 유출량 보정을 위해 보령댐의 실 측 방류량을 이용하여 댐 운영모의를 고려하였고 유입량 및 방류량 자료를 활용하여 모형의 보정(2007~2010)과 검증(2010~2016)을 실시하였다. 기후변화를 반영하기 위해 APCC의 26개 CMIP5 GCM 자료 중 RCP (Representative Concentration Pathway)4.5와 RCP 8.5 시나리오를 SPI와 극한 가뭄지수로 분석하여 RCP 8.5 BCC-CSM1-1-M을 극한 가뭄 시나리오로 선정하였다. 2005년부터 2016년까지의 일별 관측자료로 다중회귀분석하여 월별 방류량 추정식을 만들었고, 1월부터 12월까지 각 식들의 결정계수 R2는 0.57 이상으로 나타났다. 선정된 극한 가뭄 시나리오 기상자료를 방류량 추정식에 대입하여 미래기간 일별 방류량을 구축하였다. SWAT 수문평가 결과, S3 (2037~2046) 기간 봄철 저수량이 34.0% 감소하는 것으로 분석되었다. Runs 이론을 바탕으로 물부족의 심도를 구한 다음 재현기간에 따른 빈도해석을 하였다. 5~10년 빈도의 심 도로 발생하는 물부족이 미래기간에 발생하는 빈도로 보령댐의 물부족을 평가하였다. 물부족 평가 결과, S3 (2037~2046) 기간에서 5~10년 빈도의 심도를 가지는 물부족이 기준기간(2007~2016) 보다 2회 더 발생하였으며 S3 (2037~2046)에 물부족 계획 수립이 필요하다고 판단하였다.

본 연구의 목적은 기상자료(강수량, 최고기온, 최저기온, 평균기온, 평균풍속) 기반의 다중선형 회귀모형을 개발하여 농업용저수지 저수율을 예측 하는 것이다. 나이브 베이즈 분류를 활용하여 전국 1,559개의 저수지를 지리형태학적 제원(유효저수량, 수혜면적, 유역면적, 위도, 경도 및 한발빈도)을 기준으로 30개 군집으로 분류하였다. 각 군집별로, 기상청 기상자료와 한국농어촌공사 저수지 저수율의 13년(2002~2014) 자료를 활용하여 월별 회귀모형을 유도하였다. 저수율의 회귀모형은 결정계수(R2)가 0.76, Nash-Sutcliffe efficiency (NSE)가 0.73, 평균제곱근오차가 8.33%로 나타났다. 회귀모형은 2년(2015~2016) 기간의 기상청 3개월 기상전망자료인 GloSea5 (GS5)를 사용하여 평가되었다. 현재저수율과 평년저수율에 의해 산정되는 저수지 가뭄지수(Reservoir Drought Index, RDI)에 의한 ROC (Receiver Operating Characteristics) 분석의 적중률은 관측값을 이용한 회귀식에서 0.80과 GS5를 이용한 회귀식에서 0.73으로 나타났다. 본 연구의 결과를 이용해 미래 저수율을 전망하여 안정적인 미래 농업용수 공급에 대한 의사결정 자료로 사용할 수 있을 것이다.

Background : This experiment was conducted to improve the farm income by decreasing the rate of corrosion and increasing the emergence rate by selecting the proper storage method of the medicinal crop seed root. Methods and Result : Ten kinds of medicinal crop seed roots of Glycyrrhiza uralensis Fischer, Salvia miltiorrhiza Bunge, Codonopsis lanceolata, Platycodon grandiflorum, Atractylodes japonica, Angelica acutiloba, Adenophora triphylla, Rehmannia glutinosa, Angelica gigas and Cnidium officinale were used as test materials. In experiment 1, storage experiments were carried out using four treatments: untreated (dry in advance), untreated, filler (dry in advance) and filler. Drying in advance was carried for 2 days in dark place and the filler was vermiculite. For storage, in styrofoam box, seed roots were laid down in 3 replicates of 10 sets, stored in – 1℃ warehouse. The storage temperature was set as long as each seed root was not frozen investigating freezing point, supercooling point. As a result, the rate of corrosion was significantly lower in the 4th treatments (filler), such as A. japonica (30%) and A. gigas (10%), compared with untreated one. Therefore, in experiment 2, six treatments were carried out using filler. There were 6 treatments: filler disinfectant (dry in advance), filler disinfectant, filler biochar (dry in advance), filler biochar, filler disinfectant biochar (dry in advance) and filler disinfectant biochar. The disinfectant was used by metalaxyl-M at a concentration of 0.4 g/ℓ, and a biochar was used at a volume ratio of 50% (v/v). As a result, the rate of corrosion was significantly lower in the 6th treatment (filler disinfectant biochar), such as S. miltiorrhiza (13.3%) and C. lanceolata (6.7%). Conclusion : In storing medicinal crop root, it is better to store using fillers. It was shown that a mixture of filler and disinfectant biochar reduced the rate of corrosion.

흡연 유무의 남성을 대상으로 뇌 회백질의 손상 유무를 파악 할 수 있는 확산텐서영상을 검사하여 영상을 획득 한 후 Tract-Based Spatial Statics(TBSS)방법으로 뇌 회백질 부위의 기저핵 신경섬유로의 비등방도 FA(fractional anisotropy)값을 측정 분석한 결과 모든 영역에서 흡연자가 비흡연자보다 비등방성 측정값이 낮게 관찰되었으며 FA값은 통계적으로 유의하였다. 본 연구의 측정한 FA결과 값으로 추측하자면 즉, 흡연이 뇌 회백질 기저핵의 모든 해부학적 미세 구조성 변화에 크게 영향을 미치며 신경 섬유로를 손상시키고 이와 관련된 기능적 이상에 영향을 준다고 할 수 있다.

The appropriate soil depth for each species should be determined by taking into account the visual quality of the plant, including the growth conditions of the trees and total photosynthesis. The purpose of this study was to investigate the growth characteristics -height, root collar diameter, dry weight- and quality index according to soil depth. Therefore this study proposed suitable plants according to container size (soil depth) when creating a container garden. The result are as follows; Pinus densiflora, Pinus parviflora and Nandina domestica have poor growth and low ornamental value at 15 cm depth. Therefore, theu will be available at a minimum depth of 20c m. Thuja occidentalis grew in 15 cm of soil depth and had a normal visual value, but it was desirable to plant 20 cm of soil depth. Ilex serrata grew at least 20 cm above depth but it was not suitable for container garden because of its low value of ornamental value. Syringa vulgaris had little difference in growth depending on soil depth. Considering the visual quality, it will be possible to plant for container gardening at a soil depth of 20 cm or more. Container gardens are an alternative to forming green spaces in places where planting is difficult in the city. Therefore, appropriate containers and plants should be selected.

The objective of this study was to investigate the current situations of farms in South Korea which provide care farming activities in order to develop and settle care farms. A questionnaire that includes 60 questions about resources, clients, management, background of establishment, budget, manpower, organization, etc. was developed by the researchers. The survey was conducted by email and phone interview in the period of July to August, 2016. Five farms were participated in this study. As the results, each farm had a different size of land, about 744 – 39,670 m2. The farms were classified by different types of farming activities such as cultivating horticultural crops or cultivating horticultural crops and raising animals. The background of farmers was health, special education, social welfare or professional farmer. The income sources were from care farming programs, educational program, and crop production. The participants in the care farm were children with or without disability, adolescents, adolescents with behavior problem, adults, adults with mental disability or developmental disability, mental illness, dementia, addict, etc. The major activities in care farms were plant cultivating, harvesting, garden maintenance, cooking by using harvest, crop processing, waling in the farm, caring for animals, dealing with farm work equipment, and farming activity with local residents, etc. The participation duration was 25% for one-day experience, less than two weeks in a week, and more than six months, respectively. To settle and develop care farming of South Korea, the concept, clients, operation, professional manpower, policy, support, etc. should be established.

The Purpose of this study is investigated to education program and organization in Korea agricultural extension service center. In most cases, the program has steadily operated from 2014 to 2016 in metropolitan cities, specially, In Busan, the greatest education program has been set up twenty one classes during 2014~2016. This study surveyed 114 departments of 3 Agricultural Research & Extension Services and 75 Agricultural Technology Centers in 8 metropolitan cities, 9 provinces and 67 cities and counties, which implemented the consumer horticultural education programs in 2016. Twenty one agricultural extension service centers has started the consumer horticulture program undertaking for the first time in 2016. Experienced education program had been conducted to the facilities for the consumer horticulture programs in the agricultural extension service centers and training in the educational farm. Specially The Seoul Metropolitan Government had the largest educational training center in 53.136 m2, with the largest educational farm in Gangwon-do province and the most popular crops were fruit. The research suggested that the consumer horticultural of educational practice and the short & long term of improvement efforts of latent educational curriculum. Agricultural Technology Centers had lands and facilities for exiting agricultural education and the environment to use nearby farms as education farms by establishing a network. Furthermore reconsidering is needed to improve the content, a systematic study of educational practice reformation plan to improve the quality of educational practice.

Background : Heat stress induced from high temperature are known to crucially affecting on physiological properties and yield in Cnidium officinale. Methods and Results : The effect of foliar application of mixture including a urea, ascorbic acid and calcium chloride on high temperature injury of Cnidium officinale. Photosynthesis and leaf temperature in Cnidium officinale were investigated after cultivating for 24 hours at 35℃. Net photosyntheis rate, transpiration was measured at 1,000 μmol m-2 s-1 of photon flux density and leaf temperature was analyzed by thermal image. Net photosyntheis rate, stomatal conductance and transpiration rate in mixture traetment were 2 times of higher than in control. Water use efficience was not different significantly. Leaf temperature was lower in mixture treatment (25.3℃) than in control (29.0℃). Conclusion : This result show that foliar application of urea, ascorbic acid and calcium chloride was reducing a high temperature injury through a improving photosynthetical capacity and decreasing leaf temperature of Cnidium officinale.

Background : Management of air temperature are known to primarily affecting on physiological properties and yield in plant. Methods and Results : The effect of air temperature on characteristics of photosynthesis and chlorophyll fluorescence in Cnidium officinal were investigated using growth chamber after cultivating for 24 hours under controlled condition. Net photosyntheis rate, transpiration was measured at 1,000 μmol m-2 s-1 of photon flux density and chlorophyll fluorescence was analyzed by OJIP method. Net photosyntheis rate was highest in treatment of 25℃. Although transpiration rate was lowest, water use efficience was also in treatment of 25℃. Stomatal conductance was mainly influenced from ambient climatric factors such as vapor pressure deficit. As results of chlorophyll fluorescence by OJIP analysis, maximum quantum yield (Fv/Fm) of photosystem II (PSII), PIabs and the relative activities per reaction center such as ABS/RC, DIo/RC were not changed at air temperature. Therefore, elevated air temperatue during short term influence the dark reaction in photosystem through controlling a water use efficience and transpiration. Conclusion : This result show that 25℃ of air temperature may be a adequate temperature to improving the efficiency of photosynthesis in Cnidium officinale.

Background : Methicillin-Resistant Staphylococcus aureus (MRSA) is a multidrug-resistant (MDR) strain. Especially, MRSA is developing resistance to available antibacterial agents and causing complications in the treatment of infections related to skin, soft tissue, respiratory, bone, joint, and endovascular disorders. Therefore, antibacterial agent combination therapy appears to be a useful option, particularly in developing countries where antibiotic availability is limited. (+)-Usnic acid (UA) is uniquely found in lichens, and is especially abundant in genera such as Usnea and Cladonia. UA has antimicrobial activity against human and plant pathogens. Therefore, UA may be a good antibacterial drug candidate for clinical development. Methods and Results : In search of a natural products capable of inhibiting this multidrug-resistant bacteria, we have investigated the antimicrobial activity of UA against MRSA. In this study, the effects of a combination of UA and permeable agents against MRSA were investigated. For the measurement of cell wall permeability, UA with concentration of Ethylenediaminetetraacetic acid (EDTA) was used. In the other hand, Sodium azide (NaN3) was used as inhibitors of ATPase. These results suggest that the antibacterial effect of UA was potentiated by membrane-binding agents and ABC transporter-inhibiting agents, implying that antibacterial activity is associated with damage of the cell wall and inhibition of ATPase function by UA. Conclusion : UA and in combination with EDTA and NaN3 could lead to the development of new combination antibiotics against MRSA infection. The results of this study appear to be promising, and they are expected to enhance the use of natural products as drugs.

본 연구는 초저온 액체 침지식 급속 냉동으로 동결된 돈육 등심에 적합한 급속 해동방법을 선정하고 과냉각 저장이 냉동-해동 돈육의 미생물학적, 이화학적 및 관능적 품질변화에 미치는 영향을 살펴보았다. 4와 10℃ 송풍식 해동과 4와 10℃유수식 해동은 냉동 돈육 시료가 해동이 완료되는 약 290-750분 소요되었지만 27.12 MHz 라디오파 해동은 약 20분으로 가장 신속하게 돈육을 해동시켜 저장실험에 필요한 냉동 돈육의 급속 해동방법으로 선정하였다. 한편 -1.5--5℃로 24시간 냉각 처리 후 돈육 횡단면의 미세구조 분석 결과, -1.5℃에서 냉각 처리된 시료의 표면과 중심부는 동결에 의한 조직 손상이 발생하지 않았음을 확인하여 -1.5℃를 과냉각 저장 온도로 선정하였다. 저장 중 대조구인 신선육과 비교하여 냉동-해동 처리된 돈육에서 발생한 드립감량은 유의적으로(p<0.05) 높은 경향을 보였지만 -1.5℃ 과냉각 저장이 돈육의 드립감량 증가를 억제하였다. 또한 4와 15℃ 저장과 비교하여 -1.5℃ 과냉각 저장은 대조구와 냉동-해동 처리구의 TVBN과 TBARS 함량 증가, Hunter a* 값 감소와 b* 값 증가를 억제하는 효과를 보였다. 15℃ 저장 4일 후 대조구와 냉동-해동 처리구의 총 호기성 세균수는 9 log CFU/g 이상으로 급격히 증가하였다. 반면 -1.5℃ 저장 10일 후 대조구와 냉동-해동 처리구의 총 호기성 세균수는 각각 5.62와 4.43 log CFU/g으로 관찰되었다. -1.5℃ 저장 10일 동안 대조구와 냉동-해동 처리구의 대장균군과 효모 및 곰팡이 수는 저장 초기 수준으로 유지하거나 다소 감소하였다. 관능평가 결과에 있어서 4와 15℃ 저장에 비해 -1.5℃ 저장한 대조구와 냉동-해동 처리구는 모든 관능평가 항목에서 저장 중 유의적으로 높은 값을 유지하였다(p<0.05). 따라서 라디오파 유전가열 해동은 냉동 돈육 등심의 해동 과정 중 상전이 구간을 빠르게 통과함으로써 급속해동이 가능하였으며 -1.5℃ 과냉각 저장이 냉동-해동 처리 된 돈육에 얼음결정 형성 없이 품질 유지 및 미생물 생장지연에 효과적인 것을 확인하였다.

Background : Ginseng has been commonly used as a traditional oriental medicine for its wide spectrum of medicinal properties, including anti-inflammatory, antitumorigenic, adaptogenic and anti-aging properties. 20(S)-Protopanaxadiol (PPD), a intestinal metabolite of ginsenosides, is one of the active ingredients in ginseng. In this study, we have found inflammation-related genes regulated by 20(S)-PPD in mouse bone marrow-derived macrophage (BMDM) to elucidate the role of 20(S)-PPD in inflammatory signaling pathways. Methods and Results : We examined cell viability of BMDM cells after treatment of 20(S)-PPD and found that 20(S)-PPD has no cytotoxicity up to 10 uM. BMDM cells treated with none or 10uM 20(S)-PPD were used for RNA extraction and microarray analysis. It was found that 2 inflammation-related genes are upregulated and 4 genes are downregulated by 20(S)-PPD. Conclusion : These results can give clues to elucidate the role of 20(S)-PPD in inflammatory signaling pathways.

Background : Despite the presence of various bioactive compounds in ginseng, there is lack of study on the variations of bioactive compounds in ginseng according to the cultivation of soil and the applied fertilizer types (or amount). Therefore, this study aims to examine the variations of 37 fatty acids (FA) and 8 vitamin E (Vit-E) vitamers in 6-year-old ginseng root cultivated in different soil types with different fertilizers regimes. Methods and Results : The profiling of 37 FAs and 8 Vit-E vitamers in 6-year-old ginseng roots was measured by gas chromatography coupled with a flame ionization detector, and then these results were statistically analyzed with chemometrics. The FA and Vit-E content in ginseng roots varied significantly with respect to soil cultivation conditions due to organic fertilizer types and amounts used. Unsaturated FA in ginseng is approximately 2.7 fold higher than the saturated FA. Linoleic, palmitic, and oleic acids were the most abundant FAs found in the ginseng roots. Also, the major Vit-E vitamer found in ginseng root is α -tocopherol. In particular, the application of rice straw compost or food waste fertilizer was increased to create nutritionally-desirable FAs and bioactive Vit-E in ginseng root. In addition, phytonutrient profiling coupled with chemometrics can be used to discriminate the cultivation conditions of ginseng. Conclusion : This preliminary study extends our understanding about the variations of FA and Vit-E in ginseng root depending on cultivation conditions. Hence, these results can be useful as basic information for reliable ginseng production containing high amounts of phytonutrients in a paddy-converted field.

Background : Ginseng, one of most famous traditional oriental medicines, has been known for a number of pharmacological properties including anti-tumor, anti-diabetic, anti-fatigue, anti-stress, anti-oxidative, and anti-aging effects. 20(S)-Protopanaxadiol (PPD), a intestinal metabolite of ginsenosides, is one of the active ingredients in ginseng. In this study, we investigated the synergistic anticancer effect of 20(S)-PPD and temozolomide (TMZ) and the mechanism of 20(S)-PPD on glioblastoma cells. Methods and Results : We examined cell viability and the morphological changes of C6 cells after treatment of 20(S)-PPD and TMZ. 20(S)-PPD showed a potent antiproliferative activity against C6 cells by triggering apoptosis. 20(S)-PPD-induced apoptosis was characterized by a dose-dependent mitochondrial damage. 20(S)-PPD and TMZ had a synergistic effect in increasing mitochondrial damage via caspase 3 activation. Conclusion : These results revealed an unexpected mechanism of 20(S)-PPD and TMZ, triggering a mitochondrial-mediated apoptosis in C6 cells. Our findings encourage further studies of 20(S)-PPD as a promising chemopreventive agent against glioblastoma.

Background : Recently, ginseng (Panax ginseng C.A. Meyer.) berry has been used as a health-promoting supplements. Also, Mulberries (Morus alba L.) fruit have been used in traditional herbal medicine to treat and prevent diabetes. In this study, we measured the cytotoxicity after fermentation of the extracts in Panax Ginseng Berry and Mulberry Fruit. Methods and Results : The extracts were prepared by decoction for 3 hours in distilled water (100 g/L). The dried extract was then dissolved in phosphate-buffered saline (PBS) in preparation for use. Cell viability was examined by an MTT assay. RAW 264.7 cells were seeded at 1 × 104/mL densities in 96-well plates. Each grouping had a non-treated group as the control. The extracts were added to each well and incubated for 24 hours at 37°C and 5% CO2. The MTT solutions (5 mg/mL) were added to each well, and the cells were cultured for another 2 hours. The supernatant was then discarded, and 100 μL of dimethyl sulfoxide was added to each well. The optical density was read at 540 nm. Conclusion : Probiotics and prebiotics modulate the composition of human and domestic animal gut microbiota. The beneficial effects may result from suppression of harmful microorganisms or stimulation of organisms which contribute in a positive way to the nutrition and health of human and domestic animal. Recently, fermentation using microorganisms for the production of more effective compounds has been extensively studied. In particular, the novel pharmacological effects of a new compound generated by fermentation have been reported. Some previous studies have demonstrated that Fermented herbal medicine extract showed better bioactivity than normal herbal Plants extract when used at the same concentration.

Background : Angelica dahurica Bentham et Hooker, Angelica gigas Nakai, Ostericum koreanum Maximowicz and Peucedanum japonicum Thumberg　are a major medicinal plant in north Geungbuk province. Using medicinal plants are impotant it`s ingredient. Dry condition and stroage method are not standard manual. The ingredient variation of dry condition and stroage method were not researched. Methods and Results : Using plant material were cutivated on Gyongsangbukdo Bonghwa area. It were studied ingredient variation after dry and storage condition by HPLC methods. Major ingredient of Angelica gigas Nakai are decurusin, decurusinangelate. Heated air bulk dry get more decursin than natuarl dry and decurusinangelate of natural bulk dry was higher than heated air bulk dry. Major ingredient of Ostericum koreanum Maximowicz are imperatorin and isoimperatorin.. Imperatorin of Ostericum koreanum was highest peak on 50℃ heated-air dry after plastic bag sorage and isoimperatorin was highest peak on 40℃ heated-air dry after mountain cultivation. Imperatorin is a major ingredient Angelica dahurica Bentham et Hooker. Heated air bulk dry get more decursin and decursinangelate than natuarl dry and small heated-air dry. Peucedanol-7o_glucoside is a major ingredient Peucedanum japonicum Thumberg. Natural bulk dry get more peucedanol-7o_glucoside than heated-air bulk dry. Conclusion : Ingredient of Angelica dahurica Bentham et Hooker, Angelica gigas Nakai, Ostericum koreanum Maximowicz are different under various cutivation, drying method, storage. Diffent Ingedients of Angelica gigas Nakai, Ostericum koreanum Maximowicz were not accord it’s optical conditon.

The wheat-rye translocations are world widely used in wheat breeding. Among the various forms of wheat-rye translocation, the 1RS (short arm of rye chromosome 1) translocations are the most widely used because of the valuable genes (e.g. biotic / abiotic resistance genes) introduced from rye. We have developed a new wheat-rye cultivar ‘TRANS’ which have freezing resistance and high yield in this study. ‘TRANS’ is a new wheat-rye translocation cultivar developed by crossing between common wheat ‘Keumkangmil’ and ‘951188-G3-G1’, a 1AL.1RS translocation derived from ‘Fleming’ with the aim of high yield and resistant genes to various unfavorable environments carried by 1RS. ‘TRNAS’ have clearly different genetic and agronomic traits to the control cultivar ‘Keumkangmil’. The heading date and maturity of ‘TRANS’ are later than that of ‘Keumkangmil’. ‘TRANS’ has 1023 spike number per square meters and grain yield of 541 kg/10a, which are higher than ‘Keumkangmil’ (904 spike number/m2 and 504 kg/10a yield). ‘TRANS’ showed winter hardiness and powdery mildew resistance in artificial infection test and field evaluation. ‘TRANS’ gives lower flour yield than ‘Keumkangmil’ but ash and protein content were similar to that of ‘Keumkangmil’. The color of flour and noodle dough of ‘TRANS’ were darker than ‘Keumkangmil’. Quality parameters related to milling, flour quality, noodle dough and end-use quality of ‘TRANS’ indicates that ‘TRANS’ is soft wheat suitable for noodle making. ‘TRANS’ can be cultivated in entire part of Korea. Registration Grant No.: 4695

This study was conducted to investigate the change in the quality of early season Fuji apples that were subjected to eight different storage conditions using the developed controlled atmosphere (CA) storage system. Early season Fuji apples grown at Mungyeong, in the South Korea, were harvested in September 2014 for storage studies. Flesh firmness, titratable acidity, total soluble solid content, weight loss, and internal browning disorder were used as quality indicators for the stored apples. Flesh firmness and weight loss were better than that of the control after 70 days of storage. However, there was no significant difference in the titratable acidity or total soluble solid content among the apple samples from eight chambers after storage. The internal browning disorder of apple samples in CA chambers occurred when exposed to 1% CO2+2% O2 and 1% CO2+0.5% O2, resulting in a higher incidence rate of 2 and 8% after 70 days of storage than that in the control. In addition, the CA storage conditions at 3, 4 and 5% CO2+2% O2 increased the rate of internal browning disorder by 26%. Therefore, CA storage can be used to maintain the quality of the apple if the optimal gas concentrations in the CA are applied to the storage strategy.

Hepatocytes and hepatic progenitors derived from human ES cells may be a useful source for clinical application. Therefore, identification and purification of these cell types would be following important issues. There are very few candidate surface markers that can be used to identify and purify hepatic progenitor cells. In addition, indocyanine-green can be uptaken by mature hepatocytes, but cannot be applied for fluorescence activated cell sorting (FACS) due to its long emission wavelength. In the present study, we tested EpCAM as a potential marker for magnetic-activated cell sorting (MACS) of hepatic progenitors and also modified indocyanine-green into fluorescent indomonocarbocyanine for FACS-mediated sorting of mature hepatocytes after differentiation of human ES cells. Hepatic progenitor cells were sorted by MACS after incubation with anti-human EpCAM antibodies. After the final differentiation, the differentiated cells and mouse primary hepatocytes (control group) were incubated with indomonocarbocyanine and were sorted by FACS. MACS and immunocytochemistry data showed that approximately 45% of differentiated cells were EpCAM-positive cells. EpCAM-positive cells expressed α-fetoprotein, FOXa2, HnF4a, and CK18. Differentiation efficiency into albumin-positive cells was significantly higher in EpCAM-positive cells, compared to EpCAM-negative cells. Importantly, indomonocarbocyanine successfully stained cells that expressed ALB. Furthermore, FACS analysis data showed that the purity of hepatocytes that expressed albumin was significantly increased after purification of indomonocarbocyanine-positive cells. Our data demonstrated that human ES cell-derived hepatic progenitors can be efficiently isolated by MACS using EpCAM antibody. In addition, we also showed that indomonocarbocyanine can be successfully used to identify and purify mature hepatocytes using FACS.

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells have received extensive attention in the development of drug screening and toxicity testing. However, it has been reported that stem cell-derived HLCs showed hepatic functions that were too limited to be of use in drug screening and toxicity testing, possibly due to the lack of sufficient intercellular communication under conventional two-dimensional (2D) culture conditions. Therefore, a 3D differentiation system may overcome the in vitro limitation of 2D culture to produce stem cell-derived hepatocytes with mature metabolic functions. In this study, the feasibility of using a silicone-based spherofilm, specifically designed to produce spherical cell clusters, to generate uniformly sized 3D hepatic spheroids from hESCs was investigated. Hepatic spheroids generated on the spherofilm showed more homogenous size and shape than those generated in conventional low-attachment suspension culture dishes. Results of immunohistochemical analysis showed that expression of the mature hepatic marker albumin (ALB) increased over time during the hepatic maturation process. Furthermore, the 3D culture system mimicked the in vivo 3D microenvironment. Laminin, which is an important component of hepatic ECM, was expressed in hepatic spheroids. The results of immunohistochemical analysis indicated that the 3D culture environment is capable of generating an in vivo-like microenvironment. In addition, quantitative PCR analysis showed that the mature hepatic marker ALB and cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A7 were expressed at higher levels in 3D culture than in 2D culture. This indicates that the 3D culture system is suitable for hepatic maturation and that our size-controlled 3D culture conditions might accelerate hepatic function. These results suggest that 3D hepatic spheroids significantly enhance metabolic maturation of hepatocytes derived from hESCs